Two questions on the subject below: Isn't there also some effect on shedding cd8 chains under certain circumstances? Also, with most people not having as many channels available as Mario, are there any CD3+ cd45- cells or would cd3 versus 8 and 4 be sufficient to define the subpopulations? Thanks Gerhard -----Original Message----- From: Mario Roederer [SMTP:Roederer@drmr.com] Sent: Wednesday, August 09, 2000 6:03 PM To: Cytometry Mailing List Subject: Re: CD4/CD8 >We are using a 4-color mix of CD45/CD3/CD4/CD8 to determine T cell subset >ratios. However our CD4 + CD8 percentages (gated on the CD45+ CD3+ cells) >do not add up to 100%. Has anyone else had these problems or have any >suggestions? Help would be appreciated - thanks! >Lisa Whyte Lisa, Sure, there are at least (generally) 10% of CD3+ cells that are double negative (CD4-CD8-), and there is a small percentage that are double positive (CD4+CD8+). You should always calculate percentages by gating on CD3+CD45+, and then viewing a 2-parameter plot of CD4 vs. CD8. The CD4 T cells are CD4+CD8-, and the CD8 T cells are CD4-CD8+. If you have a good CD8 stain, then you can also pick out two more distinct lineages (functionally distinct, too!) that are CD8-dull and either CD4+ or CD4-. Note that for most cases, a majority of CD4+CD8+ (bright CD8, that is) cells are actually dead cells that are nonspecifically binding antibody. Note that depending on the individual and on how you gate on CD3, you may be including gamma-delta T cells or not. gd T cells are typically brighter on CD3 than alpha-beta T cells, and in many in individuals comprise a second hump above the T cells. gd T cells are either CD8+ or CD8- (and a few can be CD4+). mr
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