A rapid technique for measuring calcium uptake in mitogen-induced T And B Lymphocytes

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Abstract
Procedures for lymphocyte activation and removing cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T [thymus-derived] and B[bone marrow-derived] lymphocytes. Factors such as mouse strain, lymphocyte origin and media pH were not critical to the 45Ca2+ uptake measurements. Factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures and the nature and purity of B cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate 2-fold increase in 45Ca2+ uptake (representing an influx of .apprx. 97 pmol/lymphocyte and an increase in average cellular Ca2+ of .apprx. 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T and B cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.