# Calculating cell purity after MACS enrichment - (Mar/04/2008 )

Hello,

This might be a really basic question, but I just want to make sure I'm doing this right!

I'm purifying gd T cells from splenocytes by MACS positive selection and want to calculate the purity of my prep. I do FACS with the following staining strategy:

FITC: ab TCR, GR-1, F4/80, CD11b, CD19 (this is my dump)
PE: gd TCR
PI

As an example from my FACS analysis--
from one sample, I collected 1000000 events. I gate on live cells (PI negative), yielding 203879 cells. In that gate, I gate on FITC negative cells--77803 cells. In that gate, 75513 cells are gd TCR positive (and since that's within the previous gates, those are PI-negative, Dump-negative, gd TCR positive).

Which numbers do I use to calculate the purity of my prep? My guess is-- 75513/203879 = 37% (that's gd+ cells over total live cells) Is that correct?

Thanks so much for your help!

-carillonista-

QUOTE (carillonista @ Mar 5 2008, 12:43 AM)
Hello,

This might be a really basic question, but I just want to make sure I'm doing this right!

I'm purifying gd T cells from splenocytes by MACS positive selection and want to calculate the purity of my prep. I do FACS with the following staining strategy:

FITC: ab TCR, GR-1, F4/80, CD11b, CD19 (this is my dump)
PE: gd TCR
PI

As an example from my FACS analysis--
from one sample, I collected 1000000 events. I gate on live cells (PI negative), yielding 203879 cells. In that gate, I gate on FITC negative cells--77803 cells. In that gate, 75513 cells are gd TCR positive (and since that's within the previous gates, those are PI-negative, Dump-negative, gd TCR positive).

Which numbers do I use to calculate the purity of my prep? My guess is-- 75513/203879 = 37% (that's gd+ cells over total live cells) Is that correct?

Thanks so much for your help!

That sounds right to me, but I wonder why your purity is so low?

-Clare-