I am planning an experiment where I stimulate ex vivo PBMCs in plates for seven days before analysis of the T cell proliferation. This appears to be a pretty standard experiment, however, I can't find any information about the frequency of media changes. For longer (3+ days) PBMC culturing how often do you change the media? For complete media changes what centrifuge speed/temp do you use?
Thanks!
Hi Fiona,
Without knowing the exact details of what the goal of your research is, I can say the following. We isolated monocytes from human donors and cultured them with IL4 and GM-CSF for 5 days to dendritic cells. At the beginning of our experiments, we supplemented half of the medium every 2 days. However, we found that it is possible to cultivate the cells for 5 days without changing the medium. We did this to avoid the stress of e.g. centrifugation steps. On day 5, we transferred the cells from 6-well to 24-well plates for the test, which also seemed to make sense in terms of eliminating dead cells. Afterwards the test with contact allergens or idendification of such substances.
Sample day 0 = 1x10e6 cells in RPMI 2.5ml in 6-well plates
Greetings
Jochem
Hello, Fiona!
I typically change my media every 2-3 days, but the frequency can depend on your experimental design and vary according to cell density.
This is a detailed paper that can help you: Article A T-cell-based immunogenicity protocol for evaluating human ...