Hi,
I'm trying to set an assay of cell stimulation with PHA-L and it's just won't work! I usualy take heparinized whole blood and incubate it for 24hr in 37oC 5% CO2 with 5ug/ml
PHA-L following Cytometric beads array and FACS analysis. It always looks the same as the negative control which contains no PHA-L. What could I be doing wrong? Thanks!
Hi,
it would be preferable to separate by Ficoll the mononuclear cells (PBMC) from the peripheral blood. After this, you could either purify T cells or use the PBMC as they are. The concentration of PHA you are using is actually a bit high. I use 1ug/ml and it works perfectly after 24h. Which is your read-out for T cell stimulation? Good luck,
Silvia
Hi silvia!
thanks for the answer, I actually try to immitate the QuantiFeron stimulation kit and it works on a whole blood. most of my samples has already been stimulated with the kit but now I'm trying to cut costs so I make an effort to do it myself and I rather do it on whole blood as well. I also tried stimulation with 1 and 2.5ug/ml with no success.
could heparin interfere with PHA-L stimulation? should I try PHA-P?
Miri
Hi. Miri,
usually the peripheral blood I used was also collected in heparin. However, since you are using whole blood, you could try to dilute it at least 1:1 before performing the assay. Moreover, PHA-L should work well but you could try also with PHA-M which is the mucoprotein form which beside the blastogenic activity induces also erytrocytes agglutination. Hope it will work. Good luck,
Silvia
Hi Miri, I try to proliferate T cells with PHA-P but it does not work.Did PHA-L eventually worked with your assay?
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