Re: Blasting Cells vs Doublets

Geoffrey Lyon wrote-

>I have a user that wants to investigate the properties of blasting cells.  The
>problem that she encounters is that when presenting data people ask if she has
>performed doublet discrimination, which she cannot do because she will loose
>the blasting cells.  When performing doublet discrimination by FSC-A versus
>SSC-A gated onto SSC-H vs. SSC-W the blasting cells appear elevated in SSC-W
>with the doublets.  I was wondering if anyone has figured out how to
>distinguish doublets from blasting cells.  Any insight would be appreciated.

I'll assume for the moment we're talking about T cells. If I remember 
correctly (it's been a while since I worked on lymphocyte 
activation), the initial increase in cell volume, which occurs within 
a few (4-8) hours of stimulation, is not accompanied by a substantial 
increase in macromolecular constituents of the cell, and thus 
probably reflects a change in hydration state as much as anything 
else. At this stage of the game, one would expect increased 
expression of CD69. By 8-12 hours, you should see CD98 expression, 
and increases in both RNA content (detectable with pyronin Y using 
Hoechst 33342 or other DNA dyes to block pyronin binding to RNA) and 
mitochondrial membrane potential (probably best appreciated in this 
context using very low, i.e., nanomolar, concentrations of cyanine 
dyes). Years ago, we and others did "multiparameter" analyses of 
early lymphocyte activation using two or three parameters. I'm not 
sure that anybody has looked at the process in more detail recently, 
and it should be worthwhile to do so, taking advantage of the current 
armamentarium of reagents and of modern instruments capable of 
measuring eight and more parameters.

I should also mention that there are other changes occurring in early 
lymphocyte activation that are detectable by cytometry using physical 
parameters such as AC impedance, multiangle scatter, and fluorescence 
polarization; however, these parameters are not measurable in most 
commercial fluorescence flow cytometers.

-Howard