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Human plasma-like medium improves T lymphocyte activation

ABSTRACT: T lymphocytes are critical effectors of the immune system and the ability to study their behavior in synthetic media in vitro has facilitated major discoveries in their development, function, and fate. Recently, it has been shown that the constituents in human plasma differ markedly from commonly used synthetic media and these differences have important effects on cell physiology. We therefore compared T lymphocyte activation in human plasma-like medium (HPLM) to RPMI conventional medium both with and without serum. We found that HPLM and RPMI induced vastly different transcriptional programs, especially for transcripts encoding proteins involved in activation, proliferation and metabolism, in human peripheral blood T cells after T cell receptor (TCR) stimulation. We also found that in dialyzed fetal bovine serum (dFBS), HPLM supports a stronger response to TCR engagement compared to RPMI. In reconstruction experiments, we demonstrated that this activation difference is due to RPMI being hypocalcemic relative to the in vivo milieu and that addition of calcium chloride to RPMI can improve human T lymphocyte activation. Thus, we conclude that investigators should be cognizant of differences between commonly used media formulations and the in vivo environment since this could profoundly affect their experimental results and that physiologic media is a valuable new way to study immune cells in culture

ORGANISM(S): Homo sapiens

PROVIDER: GSE135936 | GEO | 2020/01/21

REPOSITORIES: GEO

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Project description:Folate deficiency, arsenic exposure, and gamma-IR are known developmental toxicants and have carcinogenic effects. The mechanism by which IR is known, but the effect of arsenic or folate deficiency remains unclear. Their effect may be mediated by epigenetic alterations, leading to miRNA expression changes, and this experiment examined this hypothesis We used microarrays to detail the miRNA expression profiles of TK6 cells treated with 2 uM sodium arsenite for 6 days, folate-deficient media for 6 days, or 2.5 Gy IR exposure either acutely at 4 hours post exposure or long-term at 6 days post exposure, as well as requiste controls, all in biological triplicate. Keywords: exposure differences TK6, were cultured in standard RPMI 1640 (Invitrogen Inc., Gaithersburg, MD) or folate-deficient RPMI 1640 (Invitrogen). Growth media was supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin; dialyzed fetal bovine serum (Invitrogen) was added to the folate-deficient medium in order to eliminate folic acid in the serum. For controls, folate-deficiency, arsenic exposure, and 6-day ?-IR exposure groups, 106 cells were diluted into 50ml of appropriate growth media. For the 4-hour post-?-IR exposure group, 107 cells were diluted into 50ml of growth media. For the arsenic exposed group, sodium arsenite was added to the media to a concentration of 2 ?M. For the ?-IR exposure groups, cells were diluted and allowed to incubate for 4 hours prior to irradiation treatment, and were exposed at a dose rate of 86.76 rad/min to a final dose of 2.5 Gy using a Philips MGC-40 X-ray source. Following exposure, cells were returned to the incubator. After fours hours, the short-term post-??-IR exposure group was collected for RNA isolation, as well as a mock (control) group. All experimental and control conditions were performed in triplicate. For all other groups, cells were cultured for 6 days, with the media changed and renewed, with the appropriate treatment, on day 3.

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Human plasma-like medium improves T lymphocyte activation

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