Verity House software
> RE: gate-specific data cropping
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> From: VSH Tech Sup****t <T...@[EMAIL PROTECTED]
>
> Date: Fri Feb 16 2007 - 09:01:56 EST
> Stevan
> At the risk of sounding like a commercial, WinList can do exactly what
> you
> want. The problem of "sucking up air" or perhaps having a clogged
> nozzle at
> the end of a run has gotten all of us at one time or another, and then
> the
> data (most of which is probably fine) appears to be unusable. WinList
> addresses this issue with the FTIM parameter, which is basically a
> "chronology" parameter defined over 1024 channels. Each event is
> assigned
> an FTIM channel based on its order in the listmode file, channel zero
> having
> the first n/1024 events, and channel 1023 having the last n/1024
> events.
> Displaying a 2P dot plot of FTIM vs. side scatter, for example, will
> clearly
> show the aberrant events. You may then gate on the "good" events, or
> gate
> out the "bad" events. Either way, you can salvage an otherwise bad
> run.
> You can also save the gated listmode using WinList's Save Data Source
> option, and it will contain only
> the "good" events. Another feature of the Save Data Source option is
> the
> ability to set the number of events to save in the listmode file,
> which at
> least partially addresses the desire to save a larger file into
> smaller
> segments that will each have the same number of events within given
> regions,
> as you are requesting.
> Best regards,
> Don
> Donald J. Herbert
> Technical Sup****t Manager
> Verity Software House-----Original Message-----
> From: Stevan Lauriault [mailto:ste...@[EMAIL PROTECTED]
> Sent: Friday, February 09, 2007 6:24 PM
> To: cyto-inbox
> Subject: Re: gate-specific data cropping
> Thanks for your very helpful comments. I guess what I am trying to get
> at
> is this: is
> there any analysis software available that can reduce list mode data
> in a
> reverse order-specific matter (in reverse sequence)? Correct me if
> I'm
> wrong, but I believe this function would also help in the event that a
> user
> lets a sample run dry and sucks up air bubbles, or over collects their
> intended gating target (for example, in acquisition and storage, 5000
> of
> R1).
> Thanks,
> Stevan
> ---------- Original Message ----------------------------------
> From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> Date: Wed, 7 Feb 2007 16:21:28 -0800
> >On 2/7/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> >> Hi and thanks,
> >> Since we want to directly compare MFIs (in FL2) between any two
> >> subsets (R1 vs. R3)
> >that are isolated on a bivariate dot plot (FL1 vs. FL4), doing so in
> >separate sample tubes would introduce the extra probability of
> >experimental
> error between sample tubes.
> >Directly comparing the subsets from the same sample tube, and even
> >better, the same data set, would eliminate this extra unknown.
> >It doesn't necessarily eliminate the unknown, it may just keep you from
> >finding out about it. If you were to prepare one sample on Monday, see
> >significant differences, celebrate, etc and then on Wednesday prepare
> >another sample where you didn't you'd probably want to know that (and
> >figure out why). Simply taking the FACS tube off the cytometer and
> >putting it back on won't give you that information. Now, would I be
> >surprised if that actually happened in your case? Yes.
> >Would I do replicates anyway? Yes.
> >> Let us say we have acquired 100,000 total events (stained with 3
> >> fluorochromes,
> >measured in FL1, FL2, and FL4 respectively). We are using a bivariate
> >dot plot of FL1 vs. FL4 in which we are isolating 3 subsets (R1, R2,
> >and R3) based on their relative expressions of FL1 vs. FL4. We then
> >want to compare the MFIs, as measured in FL2, among these subsets.
> >Within those 100,000 total events, there are 500 of R1, 700 of R2 and
> >1200
> of R3.
> >> If we crop the 100,000 total events to 75,000 total events (from the
> >> top in
> >stack-collected data), there will become exactly 500 events in the R2
> region. If we
> crop
> >the 100,000 total events to 40,000 total events, there will become 500
> events in R3.
> >> Now the three populations; R1, R2, and R3 each have exactly 500 total
> >> events, and we
> >can directly compare MFIs among the subsets that have identical sample
> sizes. We could
> >also then say that, when comparing means between these subsets under
> >identical experimental conditions, Rx gives a consistently higher
> fluorescent signal than Ry.
> >Well, depending on what you're doing (you've never said how you intend
> >to compare means), equal sample sizes is not particularly required so
> >there's no particular reason to cut the data to obtain equal sample
> >sizes. For example, one-way ANOVA (or one-way ANOVA with repeated
> >measure in the event that you chose to do _real_ replicates), which
> >simply says "they're different" (there are other tests, variants of
> >one-sided t-tests essentially, that give you directionality statements
> >such as mu_{R_1} > mu_{R_2} ). In this case your assumptions are:
> >1. Each group is independent
> >2. Normality of group populations (or at least "close enough" to
> >Normal) 3. The _population_ variances of the dependent variable for
> >each group are equal (or pretty close). (Actually check this. It would
> >be unsurprising to encounter large differences in the population
> >variances among your different groups).
> >Like I say, it depends a bit on what you intend to do for your
> >analysis. For example, _two_-way ANOVA actually expects equal sample
> >sizes.
> >> Is this a reasonable strategy? Is there any software available that
> >> can perform this
> >function?
> >On an FCS file directly? Probably not (neither the manipulation of the
> >flow data nor the testing). Once you've got the data out of FCS form
> >and appropriately labeled with group member****p or split into separate
> >files or something similar, then any reasonable statistics package
> >(Excel is not included in the list of reasonable statistics packages)
> >can perform the statistical tests.
> >Personally, I use R (http://www.r-project.org)
for analyzing all the
> >flow data I have, but it can be intimidating for new users (though
> >extremely powerful once you learn to use it). There are two packages in
> >R for manipulating flow data at the moment (prada and rflowcyt),
> >available through the Bioconductor Project
> >(http://www.bioconductor.org)
that can actually read in and manipulate
> >FCS data directly or slightly manipulated data from say FlowJo (so you
> >can do your gating there for example). I'm also part of a group made up
> >of the people who did rflowcyt and prada that are making a combined
> >package of the best bits of both (and some other new bits) that we
> >expect to be available in the next Bioconductor release (probably
> >sometime in April).
> >Hope that helps,
> >Byron
> >> Kind Regards,
> >> Stevan Lauriault
> >> ---------- Original Message ----------------------------------
> >> From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> >> Date: Tue, 6 Feb 2007 12:17:35 -0800
> >> >Speaking as a statistician, I would not count running the same tube
> >> >3 times or simply cutting the FCS file (the two are equivalent) as
> >> >three replicates so I wouldn't do either to achieve what you want
> >> >(though I can imagine situations where you would resample to
> >> >calculate statistics). To get three replicates you would have to
> >> >prepare three separate samples---you're interested in the
> >> >variability of the mean due to experimental error as well as the
> >> >biological variability of the cells and a single sample preparation
> >> >only
> captures the latter.
> >> >On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> >> >> Dear All,
> >> >> Question:
> >> >> We are isolating leukocyte subsets to measure and compare relative
> >> >> levels of
> >expression
> >> >> of certain antigens. For example, there are three subsets within
> >> >> a bivariate dot
> >plot;
> >> >> gated on R1, R2 and R3 respectively, and we would like to
> >> >> statistically analyze the relative mean fluorescence intensities
> >> >> of a
> biomarker, among the subsets.
> >> >> To get statistically comparable sample sizes among these subsets,
> >> >> we are running
> the
> >same
> >> >> tube three times and changing the storage criteria to 500 events
> >> >> for each subset (example, the first run is 500 events of R1, the
> >> >> second 500 of R2, and the third is
> >500
> >> >> of R3). Instead of repeating the same tube three times, and
> >> >> wasting valuable
> >sample, it
> >> >> seems like a good idea to "crop" a preexisting data file of, for
> >> >> example, 100,000
> >total
> >> >> events, three times to get an identical sample size for all three
> >> >> gated subsets
> (R1,
> >R2,
> >> >> and R3).
> >> >> Scientifically, this shouldn't be a problem, since flow cytometry
> >> >> data is collected randomly within the same sample tube. Not only
> >> >> could we run one acquisition to get different monocyte subset
> >> >> populations, but also to statistically compare constant
> >numbers
> >> >> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However,
> >> >> I can see why
> >some
> >> >> scientists might be uneasy with the idea of manipulating raw
> >> >> list-mode
> data.
> >> >> However, Since the data is collected randomly from the same
> >> >> sample, doing this
> >should
> >> >> give exactly the same readings as if we just collected less
> >> >> events, since we would
> >only
> >> >> be cropping (from stack-collected data) the most recent events
> collected.
> >> >> Currently, we are acquiring 3 different data files for each tube,
> >> >> and adjusting the acquisition and storage settings for each
> >> >> acquisition. Is there a way to perform a gate-specific data
> >> >> cropping function with any current flow cytometry data analysis
> software?
> >> >> Kind Regards,
> >> >> Stevan Lauriault
> >> >> ________________________________________________________________
> >> >> Sent via the WebMail system at lauriault.com
> >> >--
> >> >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> >> >"Oook" -- The Librarian
> >> ________________________________________________________________
> >> Sent via the WebMail system at lauriault.com
> >--
> >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> >"Oook" -- The Librarian
> ________________________________________________________________
> Sent via the WebMail system at lauriault.com
> Received on Fri Feb 16 16:38:00 2007
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> 03:12:00 E
> RE: DNA analysis software
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> From: VSH Tech Sup****t <T...@[EMAIL PROTECTED]
>
> Date: Wed Feb 21 2007 - 11:29:45 EST
> Hello Ibtissam,
> ModFit LT, for PC or Mac, has advanced modeling capability for
> research
> applications in DNA cell cycle analysis. You may use any of the model
> templates the program offers, or create your own models for non-
> traditional
> analysis, including non-mammalian DNA cell cycle studies. ModFit LT
> can be
> linked to our WinList program to provide a complete cell cycle
> analysis on
> any number of sub-populations with a single click of a button.
> For an overview, visit
<http://www.vsh.com/products>http://www.vsh.com/=
products.
> Best regards,
> Don
> Donald J. Herbert
> Technical Sup****t Manager
> Verity Software House
> ________________________________
> From: Ibtissam Abdul-Jabbar [mailto:iajab...@[EMAIL PROTECTED]
> Sent: Wednesday, February 14, 2007 11:41 PM
> To: cyto-inbox
> Subject: DNA analysis software
> Dear All, before buying software to analyse DNA on PC, I would like to
> get
> your opinion. I already have ModFit for Macintosh.
> What are you using and what do you recommend for research purposes.
> Is MultiCycle Av the one of choice?
> I appreciate your comments.
> Ibtissam A Jabbar (PhD)
> Manager of the FACS facilities
> Diamantina Institute for Cancer, Immunology and Metabolic Medicine
> (DI)
> The University of Queensland
> Level 4 R Wing
> Princess Alexandra Hospital
> Ipswich Rd Buranda QLD 4102
> Australia
> Ph: 07 3240 5945
> Fax: 07 3240 5946
> Mob: 0401154744
> Received on Thu Feb 22 15:18:00 2007
> * This message: [ Message body ]
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> Mitochondrial Membrane Potential with FACSDiva"
> * In reply to: Ibtissam Abdul-Jabbar: "DNA analysis software"
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> 03:12:00 EST
> Re: gate-specific data cropping
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> From: WEHICytometry <facs_c...@[EMAIL PROTECTED]
>
> Date: Mon Feb 12 2007 - 18:25:38 EST
> Stevan,
> For what it's worth, we have a utility we call Hackit that can crop
> cells from the front or back of a FCS file ( see
http://www.wehi.edu.au/c=
ytometry/freesoftware/index.html). We use it for
> the tasks you suggest in cleaning up violated data. I don't know
> how
> useful that would be for your current purpose because it can't do
> gating; numbers clipped are total cell numbers. I guess if you knew
> the pro****tions of your gated cells you could eventually clip out
> the
> file segments you need.
> Frank Battye.
> On 10/02/2007, at 10:24 AM, Stevan Lauriault wrote:
> > Thanks for your very helpful comments. I guess what I am trying to
> > get at is this: is
> > there any analysis software available that can reduce list mode
> > data in a reverse
> > order-specific matter (in reverse sequence)? Correct me if I'm
> > wrong, but I believe this
> > function would also help in the event that a user lets a sample run
> > dry and sucks up air
> > bubbles, or over collects their intended gating target (for
> > example, in acquisition and
> > storage, 5000 of R1).
> | | << The Cytometry Laboratory
> \__/ <<<< The Walter & Eliza Hall Institute
> ------!!<<<<<< 1G Royal Parade, Parkville
> /!!\ <<<< Victoria 3050, Australia
> o !! \ << ph: 61_3_9345 2540, fax: 61_3_9347 0852
> Received on Tue Feb 13 15:18:00 2007
> * This message: [ Message body ]
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> cells"
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> Re: gate-specific data cropping
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> From: Stevan Lauriault <ste...@[EMAIL PROTECTED]
>
> Date: Fri Feb 09 2007 - 18:24:07 EST
> Thanks for your very helpful comments. I guess what I am trying to get
> at is this: is
> there any analysis software available that can reduce list mode data
> in a reverse
> order-specific matter (in reverse sequence)? Correct me if I'm wrong,
> but I believe this
> function would also help in the event that a user lets a sample run
> dry and sucks up air
> bubbles, or over collects their intended gating target (for example,
> in acquisition and
> storage, 5000 of R1).
> Thanks,
> Stevan
> ---------- Original Message ----------------------------------
> From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> Date: Wed, 7 Feb 2007 16:21:28 -0800
> >On 2/7/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> >> Hi and thanks,
> >> Since we want to directly compare MFIs (in FL2) between any two
subset=
s (R1 vs. R3)
> >that are isolated on a bivariate dot plot (FL1 vs. FL4), doing so in
sep=
arate sample
> >tubes would introduce the extra probability of experimental error
betwee=
n sample tubes.
> >Directly comparing the subsets from the same sample tube, and even
bette=
r, the same data
> >set, would eliminate this extra unknown.
> >It doesn't necessarily eliminate the unknown, it may just keep you
> >from finding out about it. If you were to prepare one sample on
> >Monday, see significant differences, celebrate, etc and then on
> >Wednesday prepare another sample where you didn't you'd probably want
> >to know that (and figure out why). Simply taking the FACS tube off the
> >cytometer and putting it back on won't give you that information. Now,
> >would I be surprised if that actually happened in your case? Yes.
> >Would I do replicates anyway? Yes.
> >> Let us say we have acquired 100,000 total events (stained with 3
fluor=
ochromes,
> >measured in FL1, FL2, and FL4 respectively). We are using a bivariate
d=
ot plot of FL1
> >vs. FL4 in which we are isolating 3 subsets (R1, R2, and R3) based on
th=
eir relative
> >expressions of FL1 vs. FL4. We then want to compare the MFIs, as
measur=
ed in FL2, among
> >these subsets. Within those 100,000 total events, there are 500 of R1,
=
700 of R2 and
> >1200 of R3.
> >> If we crop the 100,000 total events to 75,000 total events (from the
t=
op in
> >stack-collected data), there will become exactly 500 events in the R2
re=
gion. If we
> crop
> >the 100,000 total events to 40,000 total events, there will become 500
e=
vents in R3.
> >> Now the three populations; R1, R2, and R3 each have exactly 500 total
=
events, and we
> >can directly compare MFIs among the subsets that have identical sample
s=
izes. We could
> >also then say that, when comparing means between these subsets under
ide=
ntical
> >experimental conditions, Rx gives a consistently higher fluorescent
sign=
al than Ry.
> >Well, depending on what you're doing (you've never said how you intend
> >to compare means), equal sample sizes is not particularly required so
> >there's no particular reason to cut the data to obtain equal sample
> >sizes. For example, one-way ANOVA (or one-way ANOVA with repeated
> >measure in the event that you chose to do _real_ replicates), which
> >simply says "they're different" (there are other tests, variants of
> >one-sided t-tests essentially, that give you directionality statements
> >such as mu_{R_1} > mu_{R_2} ). In this case your assumptions are:
> >1. Each group is independent
> >2. Normality of group populations (or at least "close enough" to
Normal)
> >3. The _population_ variances of the dependent variable for each group
> >are equal (or pretty close). (Actually check this. It would be
> >unsurprising to encounter large differences in the population
> >variances among your different groups).
> >Like I say, it depends a bit on what you intend to do for your
> >analysis. For example, _two_-way ANOVA actually expects equal sample
> >sizes.
> >> Is this a reasonable strategy? Is there any software available that
c=
an perform this
> >function?
> >On an FCS file directly? Probably not (neither the manipulation of the
> >flow data nor the testing). Once you've got the data out of FCS form
> >and appropriately labeled with group member****p or split into separate
> >files or something similar, then any reasonable statistics package
> >(Excel is not included in the list of reasonable statistics packages)
> >can perform the statistical tests.
> >Personally, I use R (http://www.r-project.org)
for analyzing all the
> >flow data I have, but it can be intimidating for new users (though
> >extremely powerful once you learn to use it). There are two packages
> >in R for manipulating flow data at the moment (prada and rflowcyt),
> >available through the Bioconductor Project
> >(http://www.bioconductor.org)
that can actually read in and manipulate
> >FCS data directly or slightly manipulated data from say FlowJo (so you
> >can do your gating there for example). I'm also part of a group made
> >up of the people who did rflowcyt and prada that are making a combined
> >package of the best bits of both (and some other new bits) that we
> >expect to be available in the next Bioconductor release (probably
> >sometime in April).
> >Hope that helps,
> >Byron
> >> Kind Regards,
> >> Stevan Lauriault
> >> ---------- Original Message ----------------------------------
> >> From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> >> Date: Tue, 6 Feb 2007 12:17:35 -0800
> >> >Speaking as a statistician, I would not count running the same tube
3
> >> >times or simply cutting the FCS file (the two are equivalent) as
thre=
e
> >> >replicates so I wouldn't do either to achieve what you want (though
I
> >> >can imagine situations where you would resample to calculate
> >> >statistics). To get three replicates you would have to prepare three
> >> >separate samples---you're interested in the variability of the mean
> >> >due to experimental error as well as the biological variability of
th=
e
> >> >cells and a single sample preparation only captures the latter.
> >> >On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> >> >> Dear All,
> >> >> Question:
> >> >> We are isolating leukocyte subsets to measure and compare relative
=
levels of
> >expression
> >> >> of certain antigens. For example, there are three subsets within
a=
bivariate dot
> >plot;
> >> >> gated on R1, R2 and R3 respectively, and we would like to
statistic=
ally analyze the
> >> >> relative mean fluorescence intensities of a biomarker, among the
su=
bsets.
> >> >> To get statistically comparable sample sizes among these subsets,
w=
e are running
> the
> >same
> >> >> tube three times and changing the storage criteria to 500 events
fo=
r each subset
> >> >> (example, the first run is 500 events of R1, the second 500 of R2,
=
and the third is
> >500
> >> >> of R3). Instead of repeating the same tube three times, and
wastin=
g valuable
> >sample, it
> >> >> seems like a good idea to "crop" a preexisting data file of, for
ex=
ample, 100,000
> >total
> >> >> events, three times to get an identical sample size for all three
g=
ated subsets
> (R1,
> >R2,
> >> >> and R3).
> >> >> Scientifically, this shouldn't be a problem, since flow cytometry
d=
ata is collected
> >> >> randomly within the same sample tube. Not only could we run one
ac=
quisition to get
> >> >> different monocyte subset populations, but also to statistically
co=
mpare constant
> >numbers
> >> >> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However,
=
I can see why
> >some
> >> >> scientists might be uneasy with the idea of manipulating raw
list-m=
ode data.
> >> >> However, Since the data is collected randomly from the same
sample,=
doing this
> >should
> >> >> give exactly the same readings as if we just collected less
events,=
since we would
> >only
> >> >> be cropping (from stack-collected data) the most recent events
coll=
ected.
> >> >> Currently, we are acquiring 3 different data files for each tube,
a=
nd adjusting the
> >> >> acquisition and storage settings for each acquisition. Is there a
=
way to perform a
> >> >> gate-specific data cropping function with any current flow
cytometr=
y data analysis
> >> >> software?
> >> >> Kind Regards,
> >> >> Stevan Lauriault
> >> >> ________________________________________________________________
> >> >> Sent via the WebMail system at lauriault.com
> >> >--
> >> >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> >> >"Oook" -- The Librarian
> >> ________________________________________________________________
> >> Sent via the WebMail system at lauriault.com
> >--
> >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> >"Oook" -- The Librarian
> ________________________________________________________________
> Sent via the WebMail system at lauriault.com
> Received on Mon Feb 12 12:18:00 2007
> * This message: [ Message body ]
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: gate-specific data cropping
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> From: Nebe-Von-Caron, G <g.nebe-von-ca...@[EMAIL PROTECTED]
>
> Date: Thu Feb 08 2007 - 09:36:37 EST
> The number of events collected will not be the influential thing as
> with
> a minimum of 500 events you get already a damn good estimate of your
> MFI. Just as an educational exercise you should do is to look at MFI
> estimate of one population over events. It should indicate that your
> estimate if the sample mean after 100 "samples" (each cell being a
> sample on its own) is already pretty good. You might want to look at
> the
> distribution of mean fluorescence or more im****tant of single event
> residuals (distance from mean) over time to see if they show a trend
> which would indicate an unstable measurement.
> In the sample you propose you are more likely to see differences based
> on compensation variation between your 3 populations which again is
> independent of the number of events looked at if more than 500 have
> been
> measured. It would indeed be interesting to see how much variation you
> get in mfi between independent tubes as you are more likely to
> increase
> variation by the sample processing than by the measurement.
> One control for your experiment would be to test the MFI distribution
> by
> swapping fluorochromes to demonstrate that you are independent of
> compensation and to show that the change in MFI is not dependent of
> steric hindrance or FRET, depending of the experimental / instrument
> details.
> Regards
> Gerhard
> > -----Original Message-----
> > From: Stevan Lauriault [mailto:ste...@[EMAIL PROTECTED]
> > Sent: 07 February 2007 15:16
> > To: Cytometry Mailing List
> > Subject: Re: gate-specific data cropping
> > Hi and thanks,
> > Since we want to directly compare MFIs (in FL2) between any
> > two subsets (R1 vs. R3) that are isolated on a bivariate dot
> > plot (FL1 vs. FL4), doing so in separate sample tubes would
> > introduce the extra probability of experimental error between
> > sample tubes.
> > Directly comparing the subsets from the same sample tube, and
> > even better, the same data set, would eliminate this extra unknown.
> > Let us say we have acquired 100,000 total events (stained
> > with 3 fluorochromes, measured in FL1, FL2, and FL4
> > respectively). We are using a bivariate dot plot of FL1 vs.
> > FL4 in which we are isolating 3 subsets (R1, R2, and R3)
> > based on their relative expressions of FL1 vs. FL4. We then
> > want to compare the MFIs, as measured in FL2, among these subsets.
> > Within those 100,000 total events, there are 500 of R1, 700
> > of R2 and 1200 of R3.
> > If we crop the 100,000 total events to 75,000 total events
> > (from the top in stack-collected data), there will become
> > exactly 500 events in the R2 region. If we crop the 100,000
> > total events to 40,000 total events, there will become 500
> > events in R3.
> > Now the three populations; R1, R2, and R3 each have exactly
> > 500 total events, and we can directly compare MFIs among the
> > subsets that have identical sample sizes. We could also then
> > say that, when comparing means between these subsets under
> > identical experimental conditions, Rx gives a consistently
> > higher fluorescent signal than Ry.
> > Is this a reasonable strategy? Is there any software
> > available that can perform this
> > function?
> > Kind Regards,
> > Stevan Lauriault
> > ---------- Original Message ----------------------------------
> > From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> > Date: Tue, 6 Feb 2007 12:17:35 -0800
> > >Speaking as a statistician, I would not count running the
> > same tube 3
> > >times or simply cutting the FCS file (the two are
> > equivalent) as three
> > >replicates so I wouldn't do either to achieve what you want
> > (though I
> > >can imagine situations where you would resample to calculate
> > >statistics). To get three replicates you would have to prepare three
> > >separate samples---you're interested in the variability of
> > the mean due
> > >to experimental error as well as the biological variability of the
> > >cells and a single sample preparation only captures the latter.
> > >On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> > >> Dear All,
> > >> Question:
> > >> We are isolating leukocyte subsets to measure and compare relative
> > >> levels of
> > expression
> > >> of certain antigens. For example, there are three subsets
> > within a
> > >> bivariate dot
> > plot;
> > >> gated on R1, R2 and R3 respectively, and we would like to
> > >> statistically analyze the relative mean fluorescence
> > intensities of a
> > >> biomarker, among the subsets.
> > >> To get statistically comparable sample sizes among these
> > subsets, we
> > >> are running the
> > same
> > >> tube three times and changing the storage criteria to 500
> > events for
> > >> each subset (example, the first run is 500 events of R1,
> > the second
> > >> 500 of R2, and the third is
> > 500
> > >> of R3). Instead of repeating the same tube three times,
> > and wasting
> > >> valuable sample,
> > it
> > >> seems like a good idea to "crop" a preexisting data file of, for
> > >> example, 100,000
> > total
> > >> events, three times to get an identical sample size for all three
> > >> gated subsets (R1,
> > R2,
> > >> and R3).
> > >> Scientifically, this shouldn't be a problem, since flow cytometry
> > >> data is collected randomly within the same sample tube. Not only
> > >> could we run one acquisition to get different monocyte subset
> > >> populations, but also to statistically compare constant
> > numbers
> > >> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc.
> > However, I
> > >> can see why some scientists might be uneasy with the idea of
> > >> manipulating raw list-mode data.
> > >> However, Since the data is collected randomly from the
> > same sample,
> > >> doing this should give exactly the same readings as if we just
> > >> collected less events, since we would
> > only
> > >> be cropping (from stack-collected data) the most recent events
> > >> collected.
> > >> Currently, we are acquiring 3 different data files for
> > each tube, and
> > >> adjusting the acquisition and storage settings for each
> > acquisition.
> > >> Is there a way to perform a gate-specific data cropping
> > function with
> > >> any current flow cytometry data analysis software?
> > >> Kind Regards,
> > >> Stevan Lauriault
> > >> ________________________________________________________________
> > >> Sent via the WebMail system at lauriault.com
> > >--
> > >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> > >"Oook" -- The Librarian
> > ________________________________________________________________
> > Sent via the WebMail system at lauriault.com
> Received on Thu Feb 8 14:38:00 2007
> * This message: [ Message body ]
> * Next message: Byron Ellis: "Re: gate-specific data cropping"
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Correctio=
n"
> * Maybe in reply to: Stevan Lauriault: "gate-specific data
cropping=
"
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croppin=
g"
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> 03:12:00 EST
> Re: gate-specific data cropping
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> to ] [ Next in thread ]
> From: Byron Ellis <bcel...@[EMAIL PROTECTED]
>
> Date: Wed Feb 07 2007 - 19:21:28 EST
> On 2/7/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> > Hi and thanks,
> > Since we want to directly compare MFIs (in FL2) between any two
subsets=
(R1 vs. R3)
> that are isolated on a bivariate dot plot (FL1 vs. FL4), doing so in
> separate sample
> tubes would introduce the extra probability of experimental error
> between sample tubes.
> Directly comparing the subsets from the same sample tube, and even
> better, the same data
> set, would eliminate this extra unknown.
> It doesn't necessarily eliminate the unknown, it may just keep you
> from finding out about it. If you were to prepare one sample on
> Monday, see significant differences, celebrate, etc and then on
> Wednesday prepare another sample where you didn't you'd probably want
> to know that (and figure out why). Simply taking the FACS tube off the
> cytometer and putting it back on won't give you that information. Now,
> would I be surprised if that actually happened in your case? Yes.
> Would I do replicates anyway? Yes.
> > Let us say we have acquired 100,000 total events (stained with 3
fluoro=
chromes,
> measured in FL1, FL2, and FL4 respectively). We are using a bivariate
> dot plot of FL1
> vs. FL4 in which we are isolating 3 subsets (R1, R2, and R3) based on
> their relative
> expressions of FL1 vs. FL4. We then want to compare the MFIs, as
> measured in FL2, among
> these subsets. Within those 100,000 total events, there are 500 of
> R1, 700 of R2 and
> 1200 of R3.
> > If we crop the 100,000 total events to 75,000 total events (from the
to=
p in
> stack-collected data), there will become exactly 500 events in the R2
> region. If we crop
> the 100,000 total events to 40,000 total events, there will become 500
> events in R3.
> > Now the three populations; R1, R2, and R3 each have exactly 500 total
e=
vents, and we
> can directly compare MFIs among the subsets that have identical sample
> sizes. We could
> also then say that, when comparing means between these subsets under
> identical
> experimental conditions, Rx gives a consistently higher fluorescent
> signal than Ry.
> Well, depending on what you're doing (you've never said how you intend
> to compare means), equal sample sizes is not particularly required so
> there's no particular reason to cut the data to obtain equal sample
> sizes. For example, one-way ANOVA (or one-way ANOVA with repeated
> measure in the event that you chose to do _real_ replicates), which
> simply says "they're different" (there are other tests, variants of
> one-sided t-tests essentially, that give you directionality statements
> such as mu_{R_1} > mu_{R_2} ). In this case your assumptions are:
> 1. Each group is independent
> 2. Normality of group populations (or at least "close enough" to
> Normal)
> 3. The _population_ variances of the dependent variable for each group
> are equal (or pretty close). (Actually check this. It would be
> unsurprising to encounter large differences in the population
> variances among your different groups).
> Like I say, it depends a bit on what you intend to do for your
> analysis. For example, _two_-way ANOVA actually expects equal sample
> sizes.
> > Is this a reasonable strategy? Is there any software available that
ca=
n perform this
> function?
> On an FCS file directly? Probably not (neither the manipulation of the
> flow data nor the testing). Once you've got the data out of FCS form
> and appropriately labeled with group member****p or split into separate
> files or something similar, then any reasonable statistics package
> (Excel is not included in the list of reasonable statistics packages)
> can perform the statistical tests.
> Personally, I use R (http://www.r-project.org)
for analyzing all the
> flow data I have, but it can be intimidating for new users (though
> extremely powerful once you learn to use it). There are two packages
> in R for manipulating flow data at the moment (prada and rflowcyt),
> available through the Bioconductor Project
> (http://www.bioconductor.org)
that can actually read in and manipulate
> FCS data directly or slightly manipulated data from say FlowJo (so you
> can do your gating there for example). I'm also part of a group made
> up of the people who did rflowcyt and prada that are making a combined
> package of the best bits of both (and some other new bits) that we
> expect to be available in the next Bioconductor release (probably
> sometime in April).
> Hope that helps,
> Byron
> > Kind Regards,
> > Stevan Lauriault
> > ---------- Original Message ----------------------------------
> > From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> > Date: Tue, 6 Feb 2007 12:17:35 -0800
> > >Speaking as a statistician, I would not count running the same tube 3
> > >times or simply cutting the FCS file (the two are equivalent) as
three
> > >replicates so I wouldn't do either to achieve what you want (though I
> > >can imagine situations where you would resample to calculate
> > >statistics). To get three replicates you would have to prepare three
> > >separate samples---you're interested in the variability of the mean
> > >due to experimental error as well as the biological variability of
the
> > >cells and a single sample preparation only captures the latter.
> > >On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> > >> Dear All,
> > >> Question:
> > >> We are isolating leukocyte subsets to measure and compare relative
l=
evels of
> expression
> > >> of certain antigens. For example, there are three subsets within a
=
bivariate dot
> plot;
> > >> gated on R1, R2 and R3 respectively, and we would like to
statistica=
lly analyze the
> > >> relative mean fluorescence intensities of a biomarker, among the
sub=
sets.
> > >> To get statistically comparable sample sizes among these subsets,
we=
are running the
> same
> > >> tube three times and changing the storage criteria to 500 events
for=
each subset
> > >> (example, the first run is 500 events of R1, the second 500 of R2,
a=
nd the third is
> 500
> > >> of R3). Instead of repeating the same tube three times, and
wasting=
valuable
> sample, it
> > >> seems like a good idea to "crop" a preexisting data file of, for
exa=
mple, 100,000
> total
> > >> events, three times to get an identical sample size for all three
ga=
ted subsets (R1,
> R2,
> > >> and R3).
> > >> Scientifically, this shouldn't be a problem, since flow cytometry
da=
ta is collected
> > >> randomly within the same sample tube. Not only could we run one
acq=
uisition to get
> > >> different monocyte subset populations, but also to statistically
com=
pare constant
> numbers
> > >> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However,
I=
can see why
> some
> > >> scientists might be uneasy with the idea of manipulating raw
list-mo=
de data.
> > >> However, Since the data is collected randomly from the same sample,
=
doing this
> should
> > >> give exactly the same readings as if we just collected less events,
=
since we would
> only
> > >> be cropping (from stack-collected data) the most recent events
colle=
cted.
> > >> Currently, we are acquiring 3 different data files for each tube,
an=
d adjusting the
> > >> acquisition and storage settings for each acquisition. Is there a
w=
ay to perform a
> > >> gate-specific data cropping function with any current flow
cytometry=
data analysis
> > >> software?
> > >> Kind Regards,
> > >> Stevan Lauriault
> > >> ________________________________________________________________
> > >> Sent via the WebMail system at lauriault.com
> > >--
> > >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> > >"Oook" -- The Librarian
> > ________________________________________________________________
> > Sent via the WebMail system at lauriault.com
> --
> Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> "Oook" -- The Librarian
> Received on Thu Feb 8 14:58:00 2007
> * This message: [ Message body ]
> * Next message: Kathy Heel: "Sorting RGCs"
> * Previous message: Nebe-Von-Caron, G: "RE: gate-specific data
> cropping"
> * In reply to: Stevan Lauriault: "Re: gate-specific data cropping"
> * Next in thread: Nebe-Von-Caron, G: "RE: gate-specific data
croppi=
ng"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> Re: gate-specific data cropping
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ Maybe
i=
n
> reply to ] [ Next in thread ] [ Replies ]
> From: Stevan Lauriault <ste...@[EMAIL PROTECTED]
>
> Date: Wed Feb 07 2007 - 10:16:03 EST
> Hi and thanks,
> Since we want to directly compare MFIs (in FL2) between any two
> subsets (R1 vs. R3) that
> are isolated on a bivariate dot plot (FL1 vs. FL4), doing so in
> separate sample tubes
> would introduce the extra probability of experimental error between
> sample tubes.
> Directly comparing the subsets from the same sample tube, and even
> better, the same data
> set, would eliminate this extra unknown.
> Let us say we have acquired 100,000 total events (stained with 3
> fluorochromes, measured
> in FL1, FL2, and FL4 respectively). We are using a bivariate dot plot
> of FL1 vs. FL4 in
> which we are isolating 3 subsets (R1, R2, and R3) based on their
> relative expressions of
> FL1 vs. FL4. We then want to compare the MFIs, as measured in FL2,
> among these subsets.
> Within those 100,000 total events, there are 500 of R1, 700 of R2 and
> 1200 of R3.
> If we crop the 100,000 total events to 75,000 total events (from the
> top in
> stack-collected data), there will become exactly 500 events in the R2
> region. If we crop
> the 100,000 total events to 40,000 total events, there will become 500
> events in R3.
> Now the three populations; R1, R2, and R3 each have exactly 500 total
> events, and we can
> directly compare MFIs among the subsets that have identical sample
> sizes. We could also
> then say that, when comparing means between these subsets under
> identical experimental
> conditions, Rx gives a consistently higher fluorescent signal than Ry.
> Is this a reasonable strategy? Is there any software available that
> can perform this
> function?
> Kind Regards,
> Stevan Lauriault
> ---------- Original Message ----------------------------------
> From: "Byron Ellis" <bcel...@[EMAIL PROTECTED]
>
> Date: Tue, 6 Feb 2007 12:17:35 -0800
> >Speaking as a statistician, I would not count running the same tube 3
> >times or simply cutting the FCS file (the two are equivalent) as three
> >replicates so I wouldn't do either to achieve what you want (though I
> >can imagine situations where you would resample to calculate
> >statistics). To get three replicates you would have to prepare three
> >separate samples---you're interested in the variability of the mean
> >due to experimental error as well as the biological variability of the
> >cells and a single sample preparation only captures the latter.
> >On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> >> Dear All,
> >> Question:
> >> We are isolating leukocyte subsets to measure and compare relative
lev=
els of
> expression
> >> of certain antigens. For example, there are three subsets within a
bi=
variate dot
> plot;
> >> gated on R1, R2 and R3 respectively, and we would like to
statisticall=
y analyze the
> >> relative mean fluorescence intensities of a biomarker, among the
subse=
ts.
> >> To get statistically comparable sample sizes among these subsets, we
a=
re running the
> same
> >> tube three times and changing the storage criteria to 500 events for
e=
ach subset
> >> (example, the first run is 500 events of R1, the second 500 of R2,
and=
the third is
> 500
> >> of R3). Instead of repeating the same tube three times, and wasting
v=
aluable sample,
> it
> >> seems like a good idea to "crop" a preexisting data file of, for
examp=
le, 100,000
> total
> >> events, three times to get an identical sample size for all three
gate=
d subsets (R1,
> R2,
> >> and R3).
> >> Scientifically, this shouldn't be a problem, since flow cytometry
data=
is collected
> >> randomly within the same sample tube. Not only could we run one
acqui=
sition to get
> >> different monocyte subset populations, but also to statistically
compa=
re constant
> numbers
> >> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However, I
=
can see why some
> >> scientists might be uneasy with the idea of manipulating raw
list-mode=
data.
> >> However, Since the data is collected randomly from the same sample,
do=
ing this should
> >> give exactly the same readings as if we just collected less events,
si=
nce we would
> only
> >> be cropping (from stack-collected data) the most recent events
collect=
ed.
> >> Currently, we are acquiring 3 different data files for each tube, and
=
adjusting the
> >> acquisition and storage settings for each acquisition. Is there a
way=
to perform a
> >> gate-specific data cropping function with any current flow cytometry
d=
ata analysis
> >> software?
> >> Kind Regards,
> >> Stevan Lauriault
> >> ________________________________________________________________
> >> Sent via the WebMail system at lauriault.com
> >--
> >Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> >"Oook" -- The Librarian
> ________________________________________________________________
> Sent via the WebMail system at lauriault.com
> Received on Wed Feb 7 20:38:00 2007
> * This message: [ Message body ]
> * Next message: Wilson Mandala: "Re: detection of Tregs in PHA/PMA
> stimulated cultures of CD T cells"
> * Previous message: Matthew Hanson: "Unique op****tunity for the
rig=
ht
> person at the UW-Madison!"
> * Maybe in reply to: Stevan Lauriault: "gate-specific data
cropping=
"
> * Next in thread: Byron Ellis: "Re: gate-specific data cropping"
> * Reply: Byron Ellis: "Re: gate-specific data cropping"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> Re: gate-specific data cropping
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
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y
> to ] [ Next in thread ]
> From: Byron Ellis <bcel...@[EMAIL PROTECTED]
>
> Date: Tue Feb 06 2007 - 15:17:35 EST
> Speaking as a statistician, I would not count running the same tube 3
> times or simply cutting the FCS file (the two are equivalent) as three
> replicates so I wouldn't do either to achieve what you want (though I
> can imagine situations where you would resample to calculate
> statistics). To get three replicates you would have to prepare three
> separate samples---you're interested in the variability of the mean
> due to experimental error as well as the biological variability of the
> cells and a single sample preparation only captures the latter.
> On 2/5/07, Stevan Lauriault <ste...@[EMAIL PROTECTED]
> wrote:
> > Dear All,
> > Question:
> > We are isolating leukocyte subsets to measure and compare relative
leve=
ls of expression
> > of certain antigens. For example, there are three subsets within
=
a bivariate dot plot;
> > gated on R1, R2 and R3 respectively, and we would like to
statistically=
analyze the
> > relative mean fluorescence intensities of a biomarker, among the
subset=
s.
> > To get statistically comparable sample sizes among these subsets, we
ar=
e running the
> same
> > tube three times and changing the storage criteria to 500 events for
ea=
ch subset
> > (example, the first run is 500 events of R1, the second 500 of R2, and
=
the third is 500
> > of R3). Instead of repeating the same tube three times, and wasting
va=
luable sample,
> it
> > seems like a good idea to "crop" a preexisting data file of, for
exampl=
e, 100,000 total
> > events, three times to get an identical sample size for all three
gated=
subsets (R1,
> R2,
> > and R3).
> > Scientifically, this shouldn't be a problem, since flow cytometry data
=
is collected
> > randomly within the same sample tube. Not only could we run one
acquis=
ition to get
> > different monocyte subset populations, but also to statistically
compar=
e constant
> numbers
> > of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However, I
c=
an see why some
> > scientists might be uneasy with the idea of manipulating raw list-mode
=
data.
> > However, Since the data is collected randomly from the same sample,
doi=
ng this should
> > give exactly the same readings as if we just collected less events,
sin=
ce we would only
> > be cropping (from stack-collected data) the most recent events
collecte=
d.
> > Currently, we are acquiring 3 different data files for each tube, and
a=
djusting the
> > acquisition and storage settings for each acquisition. Is there a way
=
to perform a
> > gate-specific data cropping function with any current flow cytometry
da=
ta analysis
> > software?
> > Kind Regards,
> > Stevan Lauriault
> > ________________________________________________________________
> > Sent via the WebMail system at lauriault.com
> --
> Byron Ellis (byron.el...@[EMAIL PROTECTED]
)
> "Oook" -- The Librarian
> Received on Wed Feb 7 18:58:00 2007
> * This message: [ Message body ]
> * Next message: RAJA ALAMELU: "Storage of fixed cells before
> acquisition"
> * Previous message: Eric Van Buren: "Re: gate-specific data
croppin=
g"
> * In reply to: Stevan Lauriault: "gate-specific data cropping"
> * Next in thread: Stevan Lauriault: "Re: gate-specific data
croppin=
g"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> Re: gate-specific data cropping
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
repl=
y
> to ] [ Next in thread ]
> From: Eric Van Buren <eric.vanbu...@[EMAIL PROTECTED]
>
> Date: Wed Feb 07 2007 - 10:57:01 EST
> Stevan,
> It's not exactly what you're looking for, but the easiest "off the
> shelf" solution may
> be to acquire time as a parameter. Or, if your software allows, use
> the simulated time
> parameter. Gate on time empirically to produce the the desired number
> of events in
> each region.
> As you say, the sampling is random, so it shouldn't matter which "time
> slice" you choose.
> However, to keep a uniform protocol you may want to always start your
> time gate with time
> equals zero, i.e. starting at the beginning of the list mode file.
> This should minimize
> any time-related artifacts, such as sample settling, sample/sheath dye
> equilibrium,
> exposure to room light, temperature, etc.
> --Eric
> >Dear All,
> >Question:
> >We are isolating leukocyte subsets to measure and compare relative
level=
s of expression
> >of certain antigens. For example, there are three subsets within a
biva=
riate dot plot;
> >gated on R1, R2 and R3 respectively, and we would like to statistically
=
analyze the
> >relative mean fluorescence intensities of a biomarker, among the
subsets=
..
> >To get statistically comparable sample sizes among these subsets, we
are=
running the
> same
> >tube three times and changing the storage criteria to 500 events for
eac=
h subset
> >(example, the first run is 500 events of R1, the second 500 of R2, and
t=
he third is 500
> >of R3). Instead of repeating the same tube three times, and wasting
val=
uable sample, it
> >seems like a good idea to "crop" a preexisting data file of, for
example=
, 100,000 total
> >events, three times to get an identical sample size for all three gated
=
subsets (R1, R2,
> >and R3).
> >Scientifically, this shouldn't be a problem, since flow cytometry data
i=
s collected
> >randomly within the same sample tube. Not only could we run one
ac=
quisition to get
> >different monocyte subset populations, but also to statistically
compare=
constant
> numbers
> >of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However, I
ca=
n see why some
> >scientists might be uneasy with the idea of manipulating raw list-mode
d=
ata.
> >However, Since the data is collected randomly from the same sample,
doin=
g this should
> >give exactly the same readings as if we just collected less events,
sinc=
e we would only
> >be cropping (from stack-collected data) the most recent events
collected=
..
> >Currently, we are acquiring 3 different data files for each tube, and
ad=
justing the
> >acquisition and storage settings for each acquisition. Is there a way
to=
perform a
> >gate-specific data cropping function with any current flow cytometry
dat=
a analysis
> >software?
> >Kind Regards,
> >Stevan Lauriault
> >________________________________________________________________
> >Sent via the WebMail system at lauriault.com
> Eric Van Buren <eric.vanbu...@[EMAIL PROTECTED]
>
> Manager, Flow Cytometry Core Facility
> Karmanos Cancer Institute
> Detroit, Michigan, USA
> Received on Wed Feb 7 18:38:01 2007
> * This message: [ Message body ]
> * Next message: Byron Ellis: "Re: gate-specific data cropping"
> * Previous message: RAJA ALAMELU: "FACSort Macintosh conked off!"
> * In reply to: Stevan Lauriault: "gate-specific data cropping"
> * Next in thread: Byron Ellis: "Re: gate-specific data cropping"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> gate-specific data cropping
> * This message: [ Message body ] [ More options ]
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> thread ] [ Replies ]
> From: Stevan Lauriault <ste...@[EMAIL PROTECTED]
>
> Date: Mon Feb 05 2007 - 13:56:18 EST
> Dear All,
> Question:
> We are isolating leukocyte subsets to measure and compare relative
> levels of expression
> of certain antigens. For example, there are three subsets within a
> bivariate dot plot;
> gated on R1, R2 and R3 respectively, and we would like to
> statistically analyze the
> relative mean fluorescence intensities of a biomarker, among the
> subsets.
> To get statistically comparable sample sizes among these subsets, we
> are running the same
> tube three times and changing the storage criteria to 500 events for
> each subset
> (example, the first run is 500 events of R1, the second 500 of R2, and
> the third is 500
> of R3). Instead of repeating the same tube three times, and wasting
> valuable sample, it
> seems like a good idea to "crop" a preexisting data file of, for
> example, 100,000 total
> events, three times to get an identical sample size for all three
> gated subsets (R1, R2,
> and R3).
> Scientifically, this shouldn't be a problem, since flow cytometry data
> is collected
> randomly within the same sample tube. Not only could we run one
> acquisition to get
> different monocyte subset populations, but also to statistically
> compare constant numbers
> of monocytes, PMNs, lymphocytes, lymphocyte subsets, etc. However, I
> can see why some
> scientists might be uneasy with the idea of manipulating raw list-mode
> data.
> However, Since the data is collected randomly from the same sample,
> doing this should
> give exactly the same readings as if we just collected less events,
> since we would only
> be cropping (from stack-collected data) the most recent events
> collected.
> Currently, we are acquiring 3 different data files for each tube, and
> adjusting the
> acquisition and storage settings for each acquisition. Is there a way
> to perform a
> gate-specific data cropping function with any current flow cytometry
> data analysis
> software?
> Kind Regards,
> Stevan Lauriault
> ________________________________________________________________
> Sent via the WebMail system at lauriault.com
> Received on Tue Feb 6 13:18:00 2007
> * This message: [ Message body ]
> * Next message: Beverly Barton: "Re: Equipment Survey"
> * Previous message: Lora Barsky: "Re: Cell Quest pro and non
CellQu=
est
> data"
> * Next in thread: Eric Van Buren: "Re: gate-specific data
cropping"
> * Reply: Eric Van Buren: "Re: gate-specific data cropping"
> * Reply: Byron Ellis: "Re: gate-specific data cropping"
> * Maybe reply: Stevan Lauriault: "Re: gate-specific data cropping"
> * Maybe reply: Nebe-Von-Caron, G: "RE: gate-specific data
cropping"
> * Maybe reply: Stevan Lauriault: "Re: gate-specific data cropping"
> * Maybe reply: VSH Tech Sup****t: "RE: gate-specific data cropping"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: DNA analysis software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
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y
> to ] [ Next in thread ]
> From: Novo, David <david.n...@[EMAIL PROTECTED]
>
> Date: Fri Feb 16 2007 - 19:05:22 EST
> Hello Ibtissam,
> Multicycle is a very commonly used DNA analysis program. It can
> automatically detect the number of aneuploid populations that you have
> (if any) and runs them through several different models with different
> fitting parameters to help you determine which is the best one for
> your
> data. It also has a wide range of debris and aggregate compensation to
> allow it to be used with a wide variety of sample preparation methods.
> Multicycle has just been incor****ated as a plug in module to FCS
> Express, so that you get the benefits of the high end DNA analysis as
> well as the ease of use and gating/presentation abilities of FCS
> Express. It really makes DNA analysis a snap.
> There is some information available
athttp://www.denovosoftware.com/site/=
Multicycleplugin.shtml
> -Dave
> -----------------------------------
> David Novo
> President
> De Novo Software
> david.n...@[EMAIL PROTECTED]
> ________________________________
> From: Ibtissam Abdul-Jabbar [mailto:iajab...@[EMAIL PROTECTED]
> Sent: Wednesday, February 14, 2007 8:41 PM
> To: cyto-inbox
> Subject: DNA analysis software
> Dear All, before buying software to analyse DNA on PC, I would like to
> get your opinion. I already have ModFit for Macintosh.
> What are you using and what do you recommend for research purposes.
> Is MultiCycle Av the one of choice?
> I appreciate your comments.
> Ibtissam A Jabbar (PhD)
> Manager of the FACS facilities
> Diamantina Institute for Cancer, Immunology and Metabolic Medicine
> (DI)
> The University of Queensland
> Level 4 R Wing
> Princess Alexandra Hospital
> Ipswich Rd Buranda QLD 4102
> Australia
> Ph: 07 3240 5945
> Fax: 07 3240 5946
> Mob: 0401154744
> Received on Mon Feb 19 13:38:00 2007
> * This message: [ Message body ]
> * Next message: mmo...@[EMAIL PROTECTED]
"presenting flow data"
> * Previous message: Derek Davies: "Re: tecnicad el ciclo celular"
> * In reply to: Ibtissam Abdul-Jabbar: "DNA analysis software"
> * Next in thread: VSH Tech Sup****t: "RE: DNA analysis software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> DNA analysis software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ Next
in
> thread ] [ Replies ]
> From: Ibtissam Abdul-Jabbar <iajab...@[EMAIL PROTECTED]
>
> Date: Wed Feb 14 2007 - 23:41:15 EST
> Dear All, before buying software to analyse DNA on PC, I would like to
> get your opinion. I already have ModFit for Macintosh.
> What are you using and what do you recommend for research purposes.
> Is MultiCycle Av the one of choice?
> I appreciate your comments.
> Ibtissam A Jabbar (PhD)
> Manager of the FACS facilities
> Diamantina Institute for Cancer, Immunology and Metabolic Medicine
> (DI)
> The University of Queensland
> Level 4 R Wing
> Princess Alexandra Hospital
> Ipswich Rd Buranda QLD 4102
> Australia
> Ph: 07 3240 5945
> Fax: 07 3240 5946
> Mob: 0401154744
> Received on Thu Feb 15 12:38:00 2007
> * This message: [ Message body ]
> * Next message: moo...@[EMAIL PROTECTED]
"Fwd: Re: early
apoptoti=
c
> cells"
> * Previous message: FACSLAB: "Northern Flow Group (UK)"
> * Next in thread: Novo, David: "RE: DNA analysis software"
> * Reply: Novo, David: "RE: DNA analysis software"
> * Reply: VSH Tech Sup****t: "RE: DNA analysis software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> FACSCalibur - developing software for Macintosh
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ]
> From: Simon Crase <Simon.Cr...@[EMAIL PROTECTED]
>
> Date: Tue Feb 13 2007 - 16:32:34 EST
> I've developed software to analyze data from a FACSCalibur, and our
> client has asked whether we can ****t it to run on the FACSCalibur
> itself. I understand the FACSCalibur includes a Macintosh. I'm
> interested in knowing whether anyone has done this before, especially
> with Java, and what pitfalls they encountered.
> Simon A. Crase
> Invetech Pty Ltd
> Private Bag 44
> 495 Blackburn Road
> Mount Waverley Vic 3149
> Phone: 61 3 9211 7933
> Mobile: 0424 782 725
> Fax: 61 3 9211 7702 (facsimile)
> e-mail: simon.cr...@[EMAIL PROTECTED]
> ___________________________________________________________
> IM****TANT - This email and any attachments may be confidential. Any
> retransmissions, dissemination or other use of these materials by
> persons or entities other than the intended recipient is prohibited.
> If
> received in error, please contact us and delete all copies. Before
> opening or using attachments, check them for viruses and defects. Our
> liability is limited to resupplying any affected attachments. [Any
> representations or opinions expressed in this e.mail are those of the
> individual sender, and not necessarily those of Vision Systems
> Limited].
> Received on Wed Feb 14 16:18:00 2007
> * This message: [ Message body ]
> * Next message: William King: "Re: early apoptotic cells"
> * Previous message: gharago...@[EMAIL PROTECTED]
"correlation between
cel=
l
> cycle and cell proliferation, a question"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> Flow analysis software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ]
> From: Xiangle Sun <sunxian...@[EMAIL PROTECTED]
>
> Date: Wed Sep 12 2007 - 17:12:44 EDT
> Hi, Flowers,
> We just got BD LSRII flow cytometer with FACSDiva
> software. I know Flowjo is a popular software and some
> people use FlowJo instead of FACSDiva to do data
> analysis even it comes with instrument.
> My questions are:
> 1. What are the advantages of FlowJo and FACSDiva.
> Then is it worth of purchasing FlowJo to do analysis?
> 2. How about the feature of FlowJo and FACSDiva on DNA
> cycle analysis?
> 3. Are there any other better analysis softwares?
> either for multicolor phenotyping, DNA cycle or both?
> Any comments that based on your experience will be
> greatly appreciated!
> Xiangle Sun
>
_________________________________________________________________________=
__=AD=AD_________
> Take the Internet to Go: Yahoo!Go puts the Internet in your pocket:
> mail, news, photos &
> more.http://mobile.yahoo.com/go?refer=3D1GNXIC
> Received on Thu Sep 13 11:18:00 2007
> * This message: [ Message body ]
> * Next message: Jose Benito: "malaria"
> * Previous message: Ray Hester: "entire membrane fluor vs capped -
> equally bright by FACS?"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: CFSE proliferation software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ Maybe
i=
n
> reply to ]
> From: user facs_copy <facs_c...@[EMAIL PROTECTED]
>
> Date: Wed Sep 19 2007 - 02:03:28 EDT
> Michael,
> Depends what you mean by "analyze". If you mean just to extract cell
> numbers at each division state, we use our own Weasel program for that
> (seehttp://www.wehi.edu.au/cytometry/WEASELv2.html,
scroll to the
> bottom
> of the page). We also have a proliferation modelling program,
> CytonCalculator, that can take that proliferation data and fit a model
> calculation to it
(seehttp://www.wehi.edu.au/WEHI_Groups/indexworkgroups.=
php?id=3D115for the
> entry point that describes the process). CytonCalculator may be
> downloaded free. You'll find the download link on that page.
> Frank Battye.
> > From: Kalos, Michael
> > Sent: Thursday, September 13, 2007 5:39 PM
> > No doubt this has been brought up before: I am looking for software
> > recommendations to analyze data for measuring proliferation using
CFSE.
> > Our data is acquired on an FC500 and our computers are PC-based.
> | | < The Cytometry Laboratory
> \__/ <<<<< The Walter & Eliza Hall Institute
> ------!!<<<<<<<< Post Office, Royal Melbourne Hospital
> /!!\ <<<<< Victoria 3050, Australia
> o !! \ < ph: 61_3_9345 2541, fax: 61_3_9347 0852
> Received on Wed Sep 19 11:38:00 2007
> * This message: [ Message body ]
> * Next message: pearly.yong....@[EMAIL PROTECTED]
"Histogram overlay and
> ex****ting fcs 2.0 files question"
> * Previous message: Maria Laura: "RE: single cell sorting"
> * Maybe in reply to: Kalos, Michael: "RE: CFSE proliferation
softwa=
re"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: CFSE proliferation software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
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y
> to ] [ Next in thread ]
> From: <moo...@[EMAIL PROTECTED]
>
> Date: Mon Sep 17 2007 - 19:20:03 EDT
> I also agree. We do a lot of proliferation studies and have found
> that
> Proliferation Wizard on Modfit is very good.
> Jonni
> Quoting James F George <jgeo...@[EMAIL PROTECTED]
>:
> > I have found the Modfit software from Verity to be quite useful for
> > this, and it integrates with Winlist rather nicely, if you have it.
Do=
n
> > at Verity has been very helpful over the years in helping us implement
> > the software for routine analyses.
> > I highly recommend this company.
> > I have no financial interest in Verity. I am just a long-time user.
> > James F. George, PhD | Professor
> > Division of Cardiothoracic Surgery | University of Alabama at
> > Birmingham
> > 701 South 19th St. | Birmingham, AL 35294
> > Phone: (205) 934-4261 | Fax: (205) 975-0085
> > Email: jgeo...@[EMAIL PROTECTED]
<mailto:jgeo...@[EMAIL PROTECTED]
>
> > UAB HEALTH SYSTEM
> > Medicine that touches the world
> > ________________________________
> > From: Kalos, Michael [mailto:MKa...@[EMAIL PROTECTED]
> > Sent: Thursday, September 13, 2007 5:39 PM
> > To: cyto-inbox
> > Subject: RE: CFSE proliferation software
> > No doubt this has been brought up before: I am looking for software
> > recommendations to analyze data for measuring proliferation using
CFSE.
> > Our data is acquired on an FC500 and our computers are PC-based.
> > Thanks in advance,
> > Michael Kalos
> > Michael Kalos, Ph.D.
> > Director, Clinical Immunobiology Correlative Studies Laboratory
> > Division of Cancer Immunotherapeutics and Tumor Immunology
> > Division of Hematology and Hematopoietic Cell Transplantation
> > City of Hope
> > 1500 East Duarte Road,
> > Duarte, CA 91010-5004
> > mka...@[EMAIL PROTECTED]
> > Ph: 626.256.4673, ext. 62126
> > Fax: 626.301.8261
> > "EMF <COH.org>" made the following annotations.
> >
-----------------------------------------------------------------------=
-
> > ------
> > SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are
> > intended solely for the individual or entity to which they are
> > addressed. This communication may contain information that is
> > privileged, confidential, or exempt from disclosure under applicable
la=
w
> > (e.g., personal health information, research data, financial
> > information). Because this e-mail has been sent without encryption,
> > individuals other than the intended recipient may be able to view the
> > information, forward it to others or tamper with the information
withou=
t
> > the knowledge or consent of the sender. If you are not the intended
> > recipient, or the employee or person responsible for delivering the
> > message to the intended recipient, any dissemination, distribution or
> > copying of the communication is strictly prohibited. If you received
th=
e
> > communication in error, please notify the sender immediately by
replyin=
g
> > to this message and deleting the message and any accompanying files
fro=
m
> > your system. If, due to the security risks, you do not wish to receive
> > further communications via e-mail, please reply to this message and
> > inform the sender that you do not wish to receive further e-mail from
> > the sender.
> > =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D
> > =3D=3D=3D=3D=3D=3D
> --
> Received on Tue Sep 18 13:18:00 2007
> * This message: [ Message body ]
> * Next message: Bryant, Jenny (NSW): "Phenol Red & Luminex"
> * Previous message: O'Toole, P: "Hands-on Course in York 2008"
> * In reply to: James F George: "RE: CFSE proliferation software"
> * Next in thread: user facs_copy: "RE: CFSE proliferation
software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: CFSE proliferation software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
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> to ] [ Next in thread ] [ Replies ]
> From: James F George <jgeo...@[EMAIL PROTECTED]
>
> Date: Mon Sep 17 2007 - 09:37:30 EDT
> I have found the Modfit software from Verity to be quite useful for
> this, and it integrates with Winlist rather nicely, if you have it.
> Don
> at Verity has been very helpful over the years in helping us implement
> the software for routine analyses.
> I highly recommend this company.
> I have no financial interest in Verity. I am just a long-time user.
> James F. George, PhD | Professor
> Division of Cardiothoracic Surgery | University of Alabama at
> Birmingham
> 701 South 19th St. | Birmingham, AL 35294
> Phone: (205) 934-4261 | Fax: (205) 975-0085
> Email: jgeo...@[EMAIL PROTECTED]
<mailto:jgeo...@[EMAIL PROTECTED]
>
> UAB HEALTH SYSTEM
> Medicine that touches the world
> ________________________________
> From: Kalos, Michael [mailto:MKa...@[EMAIL PROTECTED]
> Sent: Thursday, September 13, 2007 5:39 PM
> To: cyto-inbox
> Subject: RE: CFSE proliferation software
> No doubt this has been brought up before: I am looking for software
> recommendations to analyze data for measuring proliferation using
> CFSE.
> Our data is acquired on an FC500 and our computers are PC-based.
> Thanks in advance,
> Michael Kalos
> Michael Kalos, Ph.D.
> Director, Clinical Immunobiology Correlative Studies Laboratory
> Division of Cancer Immunotherapeutics and Tumor Immunology
> Division of Hematology and Hematopoietic Cell Transplantation
> City of Hope
> 1500 East Duarte Road,
> Duarte, CA 91010-5004
> mka...@[EMAIL PROTECTED]
> Ph: 626.256.4673, ext. 62126
> Fax: 626.301.8261
> "EMF <COH.org>" made the following annotations.
> ------------------------------------------------------------------------
> ------
> SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are
> intended solely for the individual or entity to which they are
> addressed. This communication may contain information that is
> privileged, confidential, or exempt from disclosure under applicable
> law
> (e.g., personal health information, research data, financial
> information). Because this e-mail has been sent without encryption,
> individuals other than the intended recipient may be able to view the
> information, forward it to others or tamper with the information
> without
> the knowledge or consent of the sender. If you are not the intended
> recipient, or the employee or person responsible for delivering the
> message to the intended recipient, any dissemination, distribution or
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> communication in error, please notify the sender immediately by
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>
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D
> =3D=3D=3D=3D=3D=3D
> Received on Mon Sep 17 14:18:01 2007
> * This message: [ Message body ]
> * Next message: pauti...@[EMAIL PROTECTED]
"[RAAM '07 - Patrick
> Autissier] Race re****t, video and photos"
> * Previous message: Konz, Richard: "New England Cytometry Meeting
> Registration"
> * In reply to: Kalos, Michael: "RE: CFSE proliferation software"
> * Next in thread: moo...@[EMAIL PROTECTED]
"RE: CFSE
proliferatio=
n
> software"
> * Reply: moo...@[EMAIL PROTECTED]
"RE: CFSE proliferation
softwar=
e"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
> Subject ] [ By Author ] [ By messages with attachments ]
> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> GUAVA Files
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
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> to ] [ Next in thread ]
> From: Maria Laura <mcaba...@[EMAIL PROTECTED]
>
> Date: Fri Sep 14 2007 - 16:20:15 EDT
> Hi!
> I=B4ve run Reticulocite samples using Guava EasyCyte Citometer . I want
> to
> analyze Guava Files using Summit Software (MoFlo) and I don=B4t see the
> same
> Plots.
> Guava saves files FCS 3.0 and 2.0 . Summit can only read 2.0 but the
> Plots
> shows very different.
> I=B4ve analyzed FCS files from other different Citometers using the
> Summit
> software, and never had a problem.
> Is there any way to convert the files?
> Thanks in advance
> Laura Cabanas
> Buenos Aires
> Argentina
> Received on Mon Sep 17 13:38:00 2007
> * This message: [ Message body ]
> * Next message: Konz, Richard: "New England Cytometry Meeting
> Registration"
> * Previous message: Sawyer, Tom: "GLIIFCA GOLF OUTING"
> * In reply to: Kalos, Michael: "RE: CFSE proliferation software"
> * Next in thread: James F George: "RE: CFSE proliferation
software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: CFSE proliferation software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ In
repl=
y
> to ] [ Next in thread ] [ Replies ]
> From: Kalos, Michael <MKa...@[EMAIL PROTECTED]
>
> Date: Thu Sep 13 2007 - 18:38:56 EDT
> No doubt this has been brought up before: I am looking for software
> recommendations to analyze data for measuring proliferation using
> CFSE.
> Our data is acquired on an FC500 and our computers are PC-based.
> Thanks in advance,
> Michael Kalos
> Michael Kalos, Ph.D.
> Director, Clinical Immunobiology Correlative Studies Laboratory
> Division of Cancer Immunotherapeutics and Tumor Immunology
> Division of Hematology and Hematopoietic Cell Transplantation
> City of Hope
> 1500 East Duarte Road,
> Duarte, CA 91010-5004
> mka...@[EMAIL PROTECTED]
> Ph: 626.256.4673, ext. 62126
> Fax: 626.301.8261
> "EMF <COH.org>" made the following annotations.
>
-------------------------------------------------------------------------=
--=AD=AD---
> SECURITY/CONFIDENTIALITY WARNING: This message and any attachments
> are intended solely for the individual or entity to which they are
> addressed. This communication may contain information that is
> privileged, confidential, or exempt from disclosure under applicable
> law (e.g., personal health information, research data, financial
> information). Because this e-mail has been sent without encryption,
> individuals other than the intended recipient may be able to view the
> information, forward it to others or tamper with the information
> without the knowledge or consent of the sender. If you are not the
> intended recipient, or the employee or person responsible for
> delivering the message to the intended recipient, any dissemination,
> distribution or copying of the communication is strictly prohibited.
> If you received the communication in error, please notify the sender
> immediately by replying to this message and deleting the message and
> any accompanying files from your system. If, due to the security
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> please reply to this message and inform the sender that you do not
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=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
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> Received on Fri Sep 14 13:58:00 2007
> * This message: [ Message body ]
> * Next message: Liza Bronner: "single cell sorting"
> * Previous message: Brian Lee: "Phenol Red Interference"
> * In reply to: Jose Benito: "malaria"
> * Next in thread: Maria Laura: "GUAVA Files"
> * Reply: Maria Laura: "GUAVA Files"
> * Reply: James F George: "RE: CFSE proliferation software"
> * Maybe reply: user facs_copy: "RE: CFSE proliferation software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 -
> 03:12:00 EST
> RE: Cell cycle software
> * This message: [ Message body ] [ More options ]
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> to ]
> From: Tim Kute <tk...@[EMAIL PROTECTED]
>
> Date: Wed Sep 19 2007 - 11:24:35 EDT
> MODFIT
> -----Original Message-----
> From: Sara Bar-Yehuda [mailto:S...@[EMAIL PROTECTED]
> Sent: Tuesday, September 18, 2007 9:43 AM
> To: cyto-inbox
> Subject: Cell cycle software
> Dear All,
> We are interested in purchasing a cell cycle software. We looked at
> "MODFIT" and "MULTICYCLE". Which one will you recommend to use for
> MAC?
> Thank you in advance,
> Sara/Renana
> Sara Bar Yehuda, Ph.D.
> Can-Fite BioPharma
> 10 Bareket st.
> P.O.Box 7537
> Petach-Tikva 49170
> Israel
> Tel: 972-3-9241114
> Fax: 972-3-9249378
> E-mail: s...@[EMAIL PROTECTED]
> Received on Thu Sep 20 12:18:00 2007
> * This message: [ Message body ]
> * Next message: Sergey Dzekunov: "FACS on platelets"
> * Previous message: Andy Heath: "MDCK cells"
> * In reply to: Sara Bar-Yehuda: "Cell cycle software"
> * Contem****ary messages sorted: [ By Date ] [ By Thread ] [ By
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> 03:12:00 EST
> Cell cycle software
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ] [ Next
in
> thread ] [ Replies ]
> From: Sara Bar-Yehuda <S...@[EMAIL PROTECTED]
>
> Date: Tue Sep 18 2007 - 09:43:06 EDT
> Dear All,
> We are interested in purchasing a cell cycle software. We looked at
> "MODFIT" and
> "MULTICYCLE". Which one will you recommend to use for MAC?
> Thank you in advance,
> Sara/Renana
> Sara Bar Yehuda, Ph.D.
> Can-Fite BioPharma
> 10 Bareket st.
> P.O.Box 7537
> Petach-Tikva 49170
> Israel
> Tel: 972-3-9241114
> Fax: 972-3-9249378
> E-mail: s...@[EMAIL PROTECTED]
> * Application/Octet-stream attachment: -
> * Application/Octet-stream attachment: -
> <!-- attachment=3D"02--" -->
> Received on Tue Sep 18 14:18:00 2007
> * This message: [ Message body ]
> * Next message: Maria Laura: "RE: single cell sorting"
> * Previous message: Rachael Walker: "summary of replies:
fluorescen=
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> probes"
> * Next in thread: Tim Kute: "RE: Cell cycle software"
> * Reply: Tim Kute: "RE: Cell cycle software"
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> New Flow Cytometry Website
> * This message: [ Message body ] [ More options ]
> * Related messages: [ Next message ] [ Previous message ]
> From: Andy Riddell <Ridd...@[EMAIL PROTECTED]
>
> Date: Mon Oct 01 2007 - 04:16:59 EDT
> Hi All,
> We have developed a new Flow cytometry web site here at EMBL. The
> link is:www.fccf.embl.de/fccfweb
> Our aim is to keep this site active, that is, we want to add new
> resources/news every 2 weeks. We would very much appreciate any
> feedback or comments that you have regarding this site.
> Best Regards,
> Andy and Alexis.
> Andy Riddell
> Flow Cytometry Core Laboratory
> EMBL Heidelberg
> Meyerhofstrasse 1, 69117 Heidelberg, Germany
> Tel: +49 [0] 6221 387-8341
> Fax: +49 [0] 6221 387-8306
> -- End --
> Received on Mon Oct 1 10:58:00 2007
> * This message: [ Message body ]
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> detection"
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> 03:12:00 EST
> Cell cycle: FlowJo vs. ModFIT
> * This message: [ Message body ] [ More options ]
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> From: Marko Marjanovic <mmarj...@[EMAIL PROTECTED]
>
> Date: Mon Oct 01 2007 - 10:43:15 EDT
> Dear flowers,
> A couple of days ago I turned to FlowJo (I'm really only a beginner
> with
> this software) for some cell cycle analysis because I wanted to try
> something new (and better) than an old version of ModFIT I was
> already
> using with my FACSCalibur. Flowjo gives me more free handling in
> determining Gaussian peaks of G1 and G2 than ModFIT which is great
> for
> some really distorted histograms that I get with some treatments of
> my
> cells, where ModFIT is usually inadequate (don't know if I used the
> proper word to describe it). But then I found myself with a problem,
> or
> should I say a challenge. With ModFIT I always get the Sum of G1, S,
> G2
> phases as 100%, whether I have a lot or none <G1 and >G2 cells. In
> flowjo, if I have a histogram with a lot of dead cells (usually in my
> treated cells) the sum of the 3 phases is not a 100%, not even close,
> and thus the data analyzed this way isn't comparable to ModFIT's
> data,
> or any older data I have. The real question is what should I do now?
> Can
> a flowjo be modified so that sum would always be 100%? I cannot
> exclude
> dead cells because it is a very im****tant part of my data, and I
> don't
> want to overlook a clear error done by ModFIT in a few bad
> histograms.
> Or should I just analyze data in either one of the softwares and
> consider it a good job?
> My mind is troubled the most by the possibility that the rate of
> subG1
> cells could markedly influence the relation of the cell cycle phases.
> ??
> Thanx, Marko
> --
> Marko Marjanovic, B.Sc.
> Laboratory of Functional Genomics
> Division of Molecular Medicine
> Rudjer Boskovic Institute
> Bijenicka 54, Zagreb, HR-10000
> Phone: +385 1 4561111 (ext.1772)
> E-mail:
mmarj...@[EMAIL PROTECTED]
> Received on Tue Oct 2 11:18:00 2007
> * This message: [ Message body ]
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> Coulter Altra"
> * Previous message: Vainshtein, Inna: "RE: Alexa Fluor conjugates"
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> 03:12:00 EST
> Re: Using DIVA Exp Files in Flojo
> * This message: [ Message body ] [ More options ]
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> From: y.Isomoto <k...@[EMAIL PROTECTED]
>
> Date: Sun Oct 14 2007 - 23:50:40 EDT
> Hi Flowers
> data Inspector/Keyword OPEN
> DElete NAme SampleID&PAtentID
> ----- Original Message -----
> From: Houston, Jim
> To: Cytometry Mailing List
> Sent: Thursday, October 11, 2007 1:16 AM
> Subject: Using DIVA Exp Files in Flojo
> We have been using the BD Diva Files for some time in FloJo. I now
> have
> another problem.
> I know that DIVA uses the Specimen name when ex****ting files as FCS.
> We
> usually use the same Exp name and Specimen to make things less
> complicated.
> Now I have a set of experiments that we run with different specimen
> names
> under 1 exp. In FloJo you cannot seem to access the Specimen name
> directly.
> FloJo sup****t was unable to help in this also. BD sup****t is slow at
> best.
> If you have experience in this area please let me know some clues on
> how
> to handle this.
> I have also notice using the DIVA software that when you change some
> user
> keywords they do not ex****t out. Any idea with what is up with that?
> Still trying to get info from BD.
> All help/suggestions are appreciated.
> Jim Houston
>
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> Version: 7.5.488 / Virus Database: 269.14.8/1064 - Release Date:
> 2007/10/11 15:09
> Received on Mon Oct 15 13:58:00 2007
> * This message: [ Message body ]
> * Next message: Cyril Cohen: "Canto 2 vs. Cytomics FC500"
> * Previous message: Xincheng Zheng: "Toxicological study request"
> * In reply to: Houston, Jim: "Using DIVA Exp Files in Flojo"
> * Next in thread: FlowJo Technical Sup****t - Maciej Simm: "Re:
Usin=
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> DIVA Exp Files in Flojo"
> * Reply: FlowJo Technical Sup****t - Maciej Simm: "Re: Using DIVA
Ex=
p
> Files in Flojo"
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> Re: Using DIVA Exp Files in Flojo
> * This message: [ Message body ] [ More options ]
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> From: bunny <bu...@[EMAIL PROTECTED]
>
> Date: Mon Oct 15 2007 - 12:53:01 EDT
> Sounds like time for a DiVa listserv!
> We have been only someowhat successful maintaining keyword info on
> FSC3 ex****t of DiVa data. In some samples, the info is there, others
> it's stripped off.
> (The DiVa manual actually says this information is removed upon FSC
> ex****t). So like many, we used the TUBE info to put cryptic info :
> PT65_unstim_D3_004 etc, with the last 3 digits being the unique
> file
> number.
> Like Dr Wilson, we name the experiments BC101507 (initials & date).
> The problem we have is the WINDOWS SERVER our data travels through-
> not flowjo! If I move the data to a flashdrive & put on my mac- the
> file names are intact. If I put through the server we get BC10$f_3_#
> $005.fcs where most of the "combined" file name is stripped off.
> Now,
> if you put the files into FJ you can still use TUBE info to
> determine
> the correct filename, but its a pain.
> I have also sent a lengthy email to a BD engineer asking for help
> around this problem.
> In the meantime, a workaround (of sorts):
> label your Experiment with ONE letter. Put all your info in TUBE ID.
> Ex****t, then dump it all into a new folder that you've labelled with
> what you originally called your experiment. (Which you can rename
> after the ex****t process).
> In Flowjo, add the TUBEID to the workspace and you can use those
> keywords to sort into groups. Not the most elegant way to do
> things,
> but it streamlined data going though our servers (where we also
> backup the data!) It's also much easier than opening file FCS files
> in FJ simply ot figure out what each file contains.
> (BTW- I also found sorting your list in FJ works best if sorted by
> TIME COLLECTED since my tube info is not alphabetical)
> bunny
> =EF=BF1/4
> Bunny Cotleur, M.S.
> Lead Research Technologist, Dept of Neurosciences
> Scientific Consultant, Flow Cytometry Core
> Cleveland Clinic Foundation
> Lerner Research Institute, NC30
> 9500 Euclid Avenue
> Cleveland, OH 44195
> Lab: (216)444-1164
> *********************************************
> Caminante, no hay camino. Se hace camino al andar.
> On Oct 12, 2007, at 4:49 PM, <Anne.Wil...@[EMAIL PROTECTED]
>
> <Anne.Wil...@[EMAIL PROTECTED]
> wrote:
> > Hi to all,
> > It's interesting that this question has come up as we have some
> > related problems with our data backup of files originating from
> > DIVA. We have found that if you ex****t as FCS files all the
> > relevant info in the Exp name and file name goes with it
> > (incidentally we use the same name for each in order to keep track
> > of whose file it is and the date it was recorded). For eg. for my
> > expts the name (both Expt and specimen) is AW121007 for today -
> > 12th October 2007 (AW is my initials). So each person uses the same
> > system. If you ex****t as FCS files all this follows and analysis in
> > either FlowJo or CellQuest retains all this info. Some of the users
> > follow this with details of the expts (mouse number, timepoint
> > etc), others with just nos 1 to x......... depending on their
> > needs. Incidentally we use the same system for files generated on
> > other machines (as well as a Canto and an Aria, we currently have
> > FACScans, FACSCaliburs and believe it or not a Cyan). However, we
> > have major problems with automatic backup systems for files from
> > the Canto and Aria (ie DIVA), - if we ex****t as an 'Expt' all this
> > info is lost, the files remain a data base number which does not
> > identify the file name or even (worse) the date of acquisition,
> > just the date of ex****t. As we have around 35 users of the Canto
> > alone, this means that all the files are lumped in together with no
> > means of identifying what belongs to who other than rex****ting into
> > DIVA, and we only have this program on the cytometers themselves,
> > all our researchers use FlowJo on a site license. It is only as FCS
> > files that this information is retained.
> > So my question is - why has no one (and where are the BD software
> > engineers?) figured out a way to make this more user (and backup)
> > friendly. Not everyone has the means or even desire to use DIVA as
> > their major analysis program (as our BD technical engineer tried to
> > convince me today). OUr FACS facility manager has tried in vain to
> > get some answers from BD in vain, and our IT manager is tearing his
> > hair out.....
> > Any suggestions?
> > And is there a BD software engineer listening out there ?
> > Anne
> > Dr Anne Wilson
> > Ludwig Institute for Cancer Research
> > Lausanne Branch
> > University of Lausanne
> > CH-1066 Epalinges
> > Switzerland
> > tel: + 41 21 692 5971
> > fax: + 41 21 692 5995
> > email: Anne.Wil...@[EMAIL PROTECTED]
> > -----Original Message-----
> > From: Adrian Smith [mailto:a.sm...@[EMAIL PROTECTED]
> > Sent: vendredi, 12. octobre 2007 01:24
> > To: Cytometry Mailing List
> > Subject: Re: Using DIVA Exp Files in Flojo
> > On 11/10/2007, at 2:16 AM, Houston, Jim wrote:
> >> We have been using the BD Diva Files for some time in FloJo. I
> >> now have another problem.
> >> I know that DIVA uses the Specimen name when ex****ting files as
> >> FCS. We usually use the same Exp name and Specimen to make things
> >> less complicated.
> >> Now I have a set of experiments that we run with different
> >> specimen names under 1 exp. In FloJo you cannot seem to access
> >> the Specimen name directly. FloJo sup****t was unable to help in
> >> this also. BD sup****t is slow at best.
> >> If you have experience in this area please let me know some clues
> >> on how to handle this.
> >> I have also notice using the DIVA software that when you change
> >> some user keywords they do not ex****t out. Any idea with what is
> >> up with that?
> > Hi Jim,
> > (this is my understanding of how it works - if anyone can add or
> > change anything I would love to hear it!)
> > Diva can ex****t data in three different ways:-
> > - FCS2 - I don't know what it does with keywords here as I never
> > use it
> > - FCS3 - FCS3 files are rewritten and renamed during the ex****t
> > procedure
> > - Experiment - FCS files are moved out of the database along with
> > an xml file.
> > In both FCS3 and Experiment ex****ts it looks like the specimen name
> > is written to the "$SRC" keyword. You can use FlowJo preferences to
> > use that as part of your workspace display name and/or you can add
> > it is a column in the workspace display.
> > In the case of an Experiment ex****t you will certainly want to
> > check the preferences for display name or you may end up displaying
> > the filename which in this case is just a number (assigned by the
> > Diva database). In an FCS3 ex****t the filename is actually created
> > from a concatenation of the Specimen and Tube names.
> > When you are ex****ting as Experiment any changes in keywording made
> > after the files are recorded do not show up in the FCS files
> > themselves because the files are just moved, not rewritten. The
> > changes are included in the accompanying xml files (which is not
> > (currently?) much use in FlowJo). If you ex****t as FCS 3 the
> > changes in keywords are included in the files as they are rewritten.
> > Because of the keyword issue we have been ex****ting as FCS3 files
> > but that creates two problems - (i) it is much slower and (ii) the
> > filenames can end up longer than 28 character which can create
> > problems keeping track of them in FlowJo (Mac) if you move them
> > around after starting your analysis.
> > What would be ideal would be an ex****t mechanism that resulted in
> > updated keywords in FCS files but also retained the xml (so Diva
> > experiment specific information can be retained in case it needs to
> > be reim****ted into Diva). Ideally this could be run as a batch job
> > overnight in order to maximise analysis time during working hours.
> > Anyone in the software group at BD listening :)
> > Regards,
> > Adrian Smith
> > Centenary Institute, Sydney, Australia
> Received on Tue Oct 16 14:58:00 2007
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> Flojo"
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