EVIDENCE FOR AN EARLY SULFHYDRYL REAGENT SENSITIVE STEP DURING LYMPHOCYTE ACTIVATION

Abstract

Publisher Summary This chapter presents evidence for an early sulfhydryl reagent sensitive step during lymphocyte activation. In a study described in the chapter, human peripheral blood lymphocytes were prepared from heparinized blood by dextran sedimentation and isopycnic centrifugation on Ficoll/Hypaque gradients. In the study, 2 × 105 lymphocytes were cultured in 0.2 ml in microtitre plates with mitogenic concentrations of phytohemagglutinin (PHA) or ConA. Diamide [diazine dicarboxylic acid bis (N,N-dimethylamide)] was added at the initiation of the culture or at various times thereafter. Activation was assayed by adding 0.1 μCi of 3H thymidine to each well at 68 h of culture, and the lymphocytes were harvested at 72 h using an automated multiple sample harvester. When diamide was added coincidentally with lectin, there was a dose-related inhibition of activation with no decrease in 3H thymidine incorporation at 10−5 M and complete suppression of activation at 10−3 M or greater. If diamide (2 × 10−4 M) was added to culture at various times after PHA or ConA, there was complete inhibition of activation up to 30 min and progressively less suppression at longer times.