Hello,
I m currently doing MLR with human PBMC and I m using CTV in order to observe the proliferation. The two first experiences worked well with a niveau proliferation with CTV. But since then attention the FACS I See the CTV but there is no more the layers of proliferation despite the fact that in culture in the 96well plate I Clearly see proliferation. I even tried with CD3/CD28 activation, with fresh and defrozed cells. Can anyone help me? My potential hypothesis is that I frozed and defrozed several times the CTV but is it the reason... Thank you
How sure are you that you are seeing proliferation in your 96-well plate wells? Dead/dying cells can clump together in a way that often look like clusters of proliferating cells but, in fact, are not. Have you been monitoring your cultures by doing cell counts? I suspect that the problem isn't the CTV reagent, but that you are actually not getting the proliferation you think you are.
T cell expansion from PBMC isn't trivial, there are a lot of things that can go wrong with it. What is your protocol?
Hello,
Thank you for your response. I'm pretty sure that I see proliferation and I confirmed it with a colleague who has experience in MLR with human PMBC. I don't monitor my culture by cells counts but visually it is growing.
For the last tests that I performed, I seeded 200 000 CTV marked PBMC per well with activation by CD3 coating or CD3/CD28 beads in complete RPMI medium. I tried with defrozed cells and fresh cells. I leave the cells for 48 to 72 hours in culture. After that I marked the cells for CD3, CD4 and CD8 and analyze with flow cytometry with also a viability marker so I know that the cells are alived.
I wouldn't expect to see very much proliferation after 72h incubation. Typically, after 2-3 days, I see blast formation rapid proliferation after that. If you extend the length of your experiment, you might start to see the signal you're looking for.