Hello!
I'd appreciate any input on the advantages/disadvantages of using a positive or negative T cell isolation kits (STEMCELL EasySep) for in vitro assays.
I would like to isolate T cells from human PBMCs and culture them under various conditions for T cell proliferation assays and cytokine production.
Should I use T cells that have been negatively selected (without any attached magnetic beads) for in vitro assays? I'm wondering if the beads from a positive selection kit (CD3+) may interfere with T cell functionality...?
I have used the negative selection kit and differentiated and did some flowcytometry on them and it worked very well. I didn't use the positive selection as I had an option. If you have an option I think you should definitely use the negative selection kit.
Is there any specific reason for you to consider the positive selection?
Thanks Sajjeev, I just happen to have a positive isolation kit available, but have not used it yet. Was debating if I should purchase a negative selection kit to maximize my gain on results rather than risking any trouble with the positive kit (although, I do wonder if the positive selection kit offers more purity than negative selection...)
Hi Frances,
if you have the opportunity, I would always go with negative selection, especially for T cells. Because of the CD3 binding, it can lead to some kind of "priming" and could falsify your results, in negative selection, the cells remain untouched. The purity depends on the principle of the isolation, of course the positive selection gives you all cells that are CD3 positive due to the specifity of the beads. the negative selection tries to separate out everything that is CD3 negative, but it is impossible to design a perfect depletion cocktail to target all cells that do not carry CD3 molecules. The difference in purity between positive selection and negative selection is roughly 99% to 95% pure, so that shouldnt be a problem.