Hello,
I want to measure the proliferation of magnetically isolated T cells. I have successfully stimulated PBMC with 5 ug/mL PHA-L and measured their proliferation with MTT. The same method has not been successful in my magnetically isolated T cells (>95% CD3+).
I would like to know if PHA-L alone will stimulate the proliferation of isolated T cells (>95% CD3+) or if an additional stimulant is required?
Thank you,
Thomas
Dear Thomas Byrne,
Yes, it is possible to stimulate the magnetically isolated T cells. You may consider using interleukin-2 (IL-2), a potent T-cell mitogenic cytokine along with PHA.
Hope this helps.
Best - Pradyumna
Induction of proliferation of very pure T cell cultures is difficult with PHA-L/IL-2 alone (Article Human T cell activation with phytohemagglutinin. The functio...
). You may want to add other cytokines (these authors suggest IL-6). While activation may occur, the magnitude will be 4-5 times lower than with APCs (Article Cellular interactions in the proliferative response of human...
).
Two possibilities:
a. Positive selection (CD3+) might interfere with subsequent PHA-stimulation. Try negative selection strategy keeping T cells untouched.
b. Missing costimulatory signal. Perform costimulatory stimulation (like antiCD28 as positive control.
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