Do I need to activate PBMC with beads or antibody before I add them to human tumor cells in 2D or 3D culture? Any protocols, experience, or references will be great!
Hello Dr. Liqiang Zhang
You need to isolate T cells from PBMCs after Ficoll-Paque density gradient centrifugation of peripheral blood and subsequently activate them. T cell activation via the αβ-T cell receptor (TCR complex) is required for in vitro expansion.
It will again depend on the aim of your experiment. In immune-oncology assays, use of pre-stimulated effector T cells with anti-CD3 and anti-CD28 antibodies, relatively high doses of cytokines (e.g., IL-2, IFNγ) or unspecific stimulation with concanavalin A or PMA and ionomycin, could bias the results when one is testing immunotherapeutic agents by generating stronger immune responses. Such assays are suited to assess the response of effector immune cells, but they cannot be optimal for the screening of immunotherapeutic agents.
So, depending on your requirements, if you would be doing immunotherapy testing, then addition of immune stimulant agents is not advisable since it could mask the results.
You could refer to the link attached below for the protocol.
https://star-protocols.cell.com/protocols/1981
Best.
If you are assessing nk cell activity against the tumor cells, then no. However, adding il-2 would increase the activity against the cell lines. Standard tumor cell lines for looking at nk cell activity include Raji and k562.
Thanks Lora. Very good points.
I am interested in the cell penetration into 3D structure, so NK, monocytes, and T cells are all important.
Hello Liqiang Zhang, I'm also interested in observing a similar phenomenon of immune cell (PBMC) penetration within spheroids. It would be highly appreciated if you could share your coculture protocol.
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