phosphlow p-STAT5
I seem to be getting high background as well as inconsistent results with p-STAT5 staining. The stim is 10 minutes with a high dose of IL2. This has been very consistent for us in a standard flow assay. We usually use thawed human PBMC and can get a 10 - 50% positivity in CD4 cells. With Mass Cyto., I'm only seeing a small shift with a 2-10 percent frequency. However, on some days it's brighter and higher. Is it necessary to fix after the intracellular stain and before the Ir labeling? Could it be from adding too many surface antibodies with the stim? Are there some tricks with mass phosphlow?
Regards,
Ian