Investigation and Standardization of the Conditions for Micro‐Lymphocyte Cultures

Abstract

Experiments were performed to establish optimal conditions for specific and non‐specific lymphocyte transformation in vitro , in cultures containing 1–3 × 10 5 human lymphocytes. The stimulation as measured by 14 C‐thymidine incorporation was evaluated when using various methods of lymphocyte purification and different numbers of contaminating erythrocytes, different kinds of tubes, serum from different sources, and varying PHA concentrations. Furthermore, the dependence of the 14 C‐thymidine incorporation on the thymidine concentration in the medium and on the total number of lymphocytes present per culture was determined. Two methods of harvesting the cells to measure the 14 C‐thymidine incorporation were compared. On the basis of this analysis, conditions for maximal sensitivity and reproducibility could be defined in measuring lymphocyte reactivity to PHA, allogeneic lymphocytes and antigens in a miniaturized culture system.