I am planning to set up a co-culture experiment involving wild type T cells, UT-T cells, with CAR-T cells derived from the same donor. The primary objective is to investigate potential allogenic reactions of the UT-T cells against against the chimeric antigen receptor (CAR) expressed by the CAR-T cells. Typically, mixed lymphocyte reaction (MLR) protocols recommend the irradiation of stimulator cells, which in my experiment would be the CAR-T cells. However, I am interested in exploring alternatives to irradiation for inhibiting T cell proliferation. Could you suggest any such methods?
You may preincubate your stimulant cells in 5x10-2 molar solution of Hydroxyurea, for 30-60' which specifically inhibits the proliferation, i.e. semiconservative DNA synthesis. This substance is nontoxic and enables the DNA-repair synthesis to proceed undisturbed in the cells. After the preincubation you have to sediment the cells and wash them with culture medium followed by viability detection
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