A simple micromethod for MLC and CML with low numbers of murine lymph node and peripheral blood lymphocytes

Abstract

A fast and simple method is described for the mixed lymphocyte culture (MLC) of low numbers of murine lymph node (LN) and peripheral blood lymphocytes (PBL). Cultures were set up with 1 X 10(4) - 4 X 10(4) responder cells in wells of inverted Terasaki plates where the cells settled at the meniscus of a hanging drop. [3H]thymidine incorporation and 51Cr-release from appropriate allogeneic target cells were determined either directly in the same hanging drop or after cell transfer and serial 2-fold dilutions in new Terasaki plates. Replicate measurements were reproducible by both methods. Responder and stimulator cell surface antigens could also be tested by addition of antisera with complement, either at the initiation or at the effector stage of culture. Since culture volumes were only 15 microliter, almost negligible quantities of antisera were required. The method was thus particularly valuable for testing with antisera of limited availability. Examples are given where inclusion of an anti-responder alloantiserum plus complement on day 0 of the MLC eliminated the day 4 cytotoxic response, and where treatment of cytotoxic effector cells with alloantiserum plus complement for 1 h prior to the 51Cr-release assay eliminated the effector cell activity.