How to determine percentage of T-cell subsets by flow. Basic math help. - (Apr/17/2009 )
I am starting a new project. Though I understand the concept I have never worked with flowcytometry before and I am very confused with how to calculate the final percentages of cells.
I have been given several flowcytometry reports and have been asked to determine the percentages of:
1. gamma-delta/ CD8 +
3. gamma-delta/ CD8 -
4. CD4-/ CD8- / gamma-delta
For some reason when I start subtracting one population from another that sounds logical to me I get results not so logical. The acquisition was done years ago and repeating it with other Ab pairings is not an option. These were the Ab pairings used:
f. CD16/56 /CD3
g. CD19/ CD3
If anyone can help me with basic math I would appreciate it.
What do you mean by your results are "not so logical"? You should be able to use the analysis software to gate the cells based on a threshold and calculate the percentages in each population automatically. What program are you using for analysis?
gfischer on Apr 17 2009, 01:27 PM said:
Thank you for your reply, gfischer!
I do not have any software. Just a bunch of printed papers with dot plots. My PI gave me an example of how to calculate CD8-/gammadelta. He suggested subtracting gamma-delta % from the CD8-/CD4- %.
No TCR 78
In the following case if I do that it will give me:
Gamma-delta/CD8- = 82% -6% = 76%
But how can there be 76% of gamma-delta/CD8- cells if 78% have no TCR?
This is what I do not find logical.
Thanks again if you can help me calculate all those subsets!