query on LPAs: replacing tritiated thymidine with flow
A.Smith at centenary.usyd.edu.AU
Mon Aug 19 08:29:57 EST 2002
At 9:35 AM -0400 9/8/02, Alice L. Givan wrote:
>ModFit software from Verity has a "Proliferation Wizard" that does
>all the proliferation
>calculations for you from cells stained with CFSE or a PKH dye (for
>gives you the precursor frequencies of the proliferating cells and
>it also gives you
>a proliferation index that tells you how many more cells you have
>now than when you
>started the culture).
>What ModFit does is model the intensity histogram of the cells ---
>so it can use the
>separate or quasi-separate peaks found in some cases with
>CFSE-stained cells. Or it
>models the theoretical positions of the peaks for dividing cells if
>you are staining
>with the PKH dyes (that don't give such good intensity separation)
>or if your CFSE
>staining hasn't given separate peaks.
I haven't ever used ModFit so I can comment on it directly, but,
FlowJo also has a proliferation platform that allows you fit curves
to CFSE-type proliferation data and generate various stats and gates.
I believe FlowJo's proliferation platform automatically takes care of
irregularities of log amps by including a parameter to the fit which
models this (I sure Mario could supply the mathematical details to
those so inclined). It can also model the contribution of
autofluorescene. Like most FlowJo analyses it all happens easily and
automatically (but you can adjust parameters manually if you need to)
and it is very easy to apply to batches of samples. Exporting to
Excel is also very easy - as already mentioned by Joanna Roberts in
this thread. I haven't quite worked out how to get it fit curves
every time but overall it seems to do pretty good job on my data.
(Disclaimer: - I provided a some input into the way the platform
works and I have been beta testing FlowJo for a while now)
BTW - after a lot of delays FlowJo 4 is coming along really nicely -
especially if you are using Mac OS X. I like the new comparison
platform a lot (it is not quite there yet but it shows lots of
potential). If you have a dual processor Mac you should check out the
latest version which has added support for multi-processors.
If you are worried about upgrading to a new dongle they have just
released v3.7 which works with both v3 and v4 dongles, just in case
you need to go back to version 3 for some reason. (Disclaimer: I also
pushed for this - there are some very conservative people in my lab
who hate software upgrades!).
Note there was another program being developed specifically for
CFSE-modelling (I think it was going to be called "CFSE Modeller").
However, it looks to have disappeared and I'm not sure what it's
status is now. I never really had the chance to test it out because
the author was overly paranoid about piracy, ie there was no way to
test it with your own data. Given that attitude it is not
particularly surprising that is failed to thrive - which is a pity
because Phil Hodgkin was involved in developing it and he has done a
LOT of mathematical modelling of CFSE-data.
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