Isaacs, Cary (CIsaacs at organo.com) wrote:
:Our lab has been trying various methods to purify
:human T cells from peripheral
:blood. We have found columns sold by Pierce to purify consistantly better then
:90% (based on flow cytometery). However, when we try to stimulate these cells
:with immobilized anti-CD3 and RPMI with 10% FCS, they will not proliferate.The
:PBMCs before the column do proliferate.The T cells also proliferate with PHA.
: Any ideas?
First, wider margins please.
Immobilized anti-CD3 will deliver signal 1 (TCR) without signal 2
(costimulation) which can lead to apoptosis and/or anergy given that
you are working with pure T cell populations.
PHA binds CD2 and delivers a signal which mimics signal 1 but may
not require the same costimulatory ligands and therefore lead to
proliferation.
I suggest adding irradiated (3000 rads) PBMCs during your anti-CD3
incubation. They will not be able to divide, so you will be left
with only live T cells. After a week the dead cells can be removed
via density gradient centrifugation. This process should give you a
4-8-fold increase in your T cells after about 10 days. However, I would
also suggest transferring the cells to a new dish (without anti-CD3)
after three days and adding IL-2 then and every 3-5 days.
Good luck.
TKendrick
