Use of [S-35]methionine-labelled rat lymphoblasts in micro-cytotoxic and limiting dilution assays

When only limited numbers of effector cells are available for in vitro T cytotoxic determinations, standard assays cannot be performed. Cr-51 is still the most commonly used marker of target cells in cytotoxicity assays but since the incorporation of this marker is low, especially in non-tumor cells such as lymphoblasts, larger numbers of both target and effector cells are required. Here we report the use of [S-35]methionine-labelled rat ConA blasts in cytotoxic, micro-cytotoxic and limiting dilution assays. Regardless of whether [S-35]methionine or Cr-51 targets were employed, cytotoxic activities were identical when large numbers of target cells (10(4)) were used. The high uptake of [S-35]methionine by ConA blasts (9 cpm/cell) permitted the use of a small number of target cells without any loss of sensitivity. Therefore, the number of effecters and targets required was dramatically reduced, especially with high E:T ratios such as 100:1. The use of low number of [S-35]methionine-labelled rat ConA blasts as targets was also suitable for the measurement of alloreactive T cell precursor frequencies. This technique illustrates the possibility of studying T cytotoxicity in animal species lacking tumor target cell lines under experimental conditions where the availability of effector cells is limited and the optimal use of such cells becomes critical.