Flow cytometry in THP-1 cells - (Oct/20/2012 )

I justed stained my THP-1 cells with Antibodies: Isotype, CD 40, CD 80, and CD 95. I incubated with the antibodies for 1hr at 4 degrees celsius.

But after analyzing with flow there was no significant fluorescence in my treatment or control cells.

I was hoping if anyone could help me, should I be incubating with the Ab for a longer duration?

Thanks.

Josh

1. How was your THP-1 cells prepared? Isolation procedure?

2. Fixative? PFA? Methanol? Ethanol?

3. Primary antibody dilution?

4. Secondary antibody dilution?

5. Would you mind attaching the final flow scatter plot (the SSC vs FSC plot and maybe a Histogram to show what you're referring to?)

6. Cell viability?