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

Abstract

When only limited numbers of effector cells are available for in vitro T cytotoxic determinations, standard assays cannot be performed. 51Cr 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 [35S]methionine-labelled rat ConA blasts in cytotoxic, micro-cytotoxic and limiting dilution assays. Regardless of whether [35S]methionine or 51Cr targets were employed, cytotoxic activities were identical when large numbers of target cells (10(4)) were used. The high uptake of [35S]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 effectors and targets required was dramatically reduced, especially with high E : T ratios such as 100 : 1. The use of low number of [35S]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.