Lactic acid treatment in T cell culture

Pan T cell isolation and activation

·        Isolate spleen

·        Isolate T cells using the Pan T cell isolation kit from Miltenyi following the manufacturer’s instructions

·        Resuspend T cells in culture medium

Set up T cell culture

·        Count T cells and adjust to 1x10⁶ T cells per 1 ml medium

·        Wash CD3/CD28 Dynabeads as described in manufacturer’s instructions and adjust to 25 µl beads for 1x10⁶ cells per 1 ml medium

·        Mix Beads with T cells and immediately seed in desired format (tested: 96 well, 24 well, 12 well, 6 well)

·        Directly add lactic acid at desired concentration (tested: 10 mM, 15 mM; stock 1 M in sterile H₂O)

·        Incubate 48 h for metabolic measurements or cell cycle analysis, 72 h for proliferation analysis

Materials:

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