I am interested in isolating effector T cells from peripheral blood. Does anyone know any recipe/kit or have any recommendation?
Do you want total effector cells? or do you want auto-aggressive effector cells? There are different protocols. For total effector do ficoll (isolates lymphocytes on the buffy coat) then do a HLA-DR (for human) MHC-II (for mouse) deplete. The remaining cells are CD4 and CD8s. You can further deplete the other population. If you want autoaggressive cells do the same steps but add a CD40 isolation at the end. CD4+CD40+ cells are autoaggressive cells.
For isolation of the whole population you can use a combination of either anti-CD4 or anti-CD8 microbeads (Milenyi) followed by depletion of the CD45RA+ subset using anti-CD45RA-Dynabeads.
For pure human T effector cell populations you should use a cell sorter (naïve (CD4+CD45RO-CD27+), central memory (CD4+CD45RO+CD27+), effector memory (CD4+CD45RO+CD27-) CD4+ T cells
Biocoll is like phicoll, is that right? If so, it is not what I had mind. I was thinking in a positive selection protocol/kit to retain activated effector T cells and leave naive T cells untouched.
Do you want total effector cells? or do you want auto-aggressive effector cells? There are different protocols. For total effector do ficoll (isolates lymphocytes on the buffy coat) then do a HLA-DR (for human) MHC-II (for mouse) deplete. The remaining cells are CD4 and CD8s. You can further deplete the other population. If you want autoaggressive cells do the same steps but add a CD40 isolation at the end. CD4+CD40+ cells are autoaggressive cells.
If you decide to isolate T cells by cell sorting or magnetic beads and thent to stimulate them, remember that you have to do a negative selection. Do not use for eg antibodies anti-CD3 to purify your T cells. They couls be already activated by the iexcess of antibodies. Then these cells could not respond to your stimulus in vitro,
Thanks a lot for all the replies. I am sorry if my question was not clear. With effector T cells I meant those that are primed and have antigen specific effector functions. As Dr. Wagner guessed, I am actually interested in isolating activated autoreactive T cells from blood, particularly CD4 but not only. I will check on the CD40 isolation protocol/kits and if someone can recommend one I will appreciate it
Hi Dr. Reche....there isnt a CD40 isolation kit. We use G28-5, the commercially available anti-CD40 and pass through the Miltenyi AutoMacs. If you do not have an AutoMAcs available, you can buy columns and a magnate system for isolation. You can check the protocol in our Clinical Immunology paper in 2007, Waid, Vaitaitis, Putnam, Gottlieb and Wagner. Also please feel free to contact me directly and I can send you the protocol. Best of luck!
I've used some time ago either Ficoll or Lymphoprep with equalizador success. But never forget to check the cell viability with Y eosin, trypan blue or another vital staining.
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