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T cell proliferation problem - (Feb/25/2011 )

Hi, everyone,
I'm analyzing CFSE T-cell proliferation after stimulation with PHA-P (5 g/ml).
The problem is that even after 72h of incubation, the significant population of cells does not proliferate (first peak on the picture).
What can be the reason for this? It is known that PHA-P has two subunits- PHA-L and PHA-E, the last one has low mitogenic activity. Would PHA-L alone result in better activation?
Attached Image

-Holsten-

Not sure about your assay but I have some experience with PHA. I have always used the L form for CD4 T cell assays. You may be below the threshold for a good T cell response using PHA-P at 5ug/ml since you don't now the level of the L form. You should try to titrate your PHA-P to see if you can find a good level for the mixed PHA. The L molecule can be had in very pure form and is quite stimulatory to CD4 T cells.

-orchidman2-

Holsten on Fri Feb 25 18:21:59 2011 said:


Hi, everyone,
I'm analyzing CFSE T-cell proliferation after stimulation with PHA-P (5 g/ml).
The problem is that even after 72h of incubation, the significant population of cells does not proliferate (first peak on the picture).
What can be the reason for this? It is known that PHA-P has two subunits- PHA-L and PHA-E, the last one has low mitogenic activity. Would PHA-L alone result in better activation?


Stimulation quality also depends on what cell population you are stimming. If it is highly purified CD4+ T cells, PHA is not great. It is known that monocytes are a crucial component to activation of PBMCs by PHA (http://www.jimmunol.org/content/122/3/1099.short). If you are using purified CD4s you need to add exogenous IL-2. I have had good responses with PHA-M. Here's more info on the PHA subunits: http://www.roche-applied-science.com/pack-insert/1082132a.pdf

-Greg S-

Hi everyone, i know its an old thread but i suffer from a similar problem.

I use 10 ug/ml PHA (L9017 Sigma) for my purified T-cells at day 0 and add 1000U/ml IL-2 at day 3. I cant detect any proliferation with my CFSE (i use 2.5uM).

any help is welcome

-Syred-