Hi,
I am new in cell culture and would like to culture human PBMCs followed by exposure to different cannabinoids.
Could anyone please advise good media, time-points and concentrations? Basically any good protocols out there?
Thank you so much in advance for all your help and advises.
Hi Carla,
A lot of groups are very interested in doing this type of study.
My question would be, what parameter of PBMC activity do you want to study, proliferation to antigen, to mitogen, cytokine synthesis in response antigen or mitogen... The culture conditions you would use for each would vary. Let me know and I will line you up with an appropriate protocol.
Randall
Hi Carla,
You can culture your PBMCs in RPMI‐1640 supplemented with 10% FBS, 100 μg/ml streptomycin, L-glutamine, and 100 UI/ml penicillin. If you want to test the effect of your molecules on polyclonal T cell proliferation it is also necessary to supplement this medium with a mitogen specific for this lymphocyte subset (e.g., 1-10 μg/ml PHA for 3-5 days). This can be done in flat bottom plates or flasks. Alternatively, you can activate T-cells with beads coated with anti-CD3/anti-CD28 (0.5–1 bead per lymphocyte), but in this case with round-wells microplates. Cell density should be 0.25-0.5 x 106 cells/mL. Then, you can use different assay readouts for cell proliferation, like dye dilutions (CFSE, CTV, CPD), labelling with 3[H]-thymidine or BrdU, MTT assay, and so on. Depending on the instruments you have access to it is probably that your culture conditions might change a little bit, like for example how long your PBMCs will be in culture.
Hope this help you
Fran
Use RPMI 20% SERUM for isolation and and 1st culture, supplement it with PHA (YOU have to standardise it), 72hrs seems to be a good time point as we estimated using kinetic blue assay
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