Sinigaglia_1983_J.Immunol.Methods_59_13

An in vitro assay which measures the proliferation response of primed murine lymph node cells of fetal calf acetylcholine receptor (AchR) is described. The character of the AChR preparation is critical. The use of reagents such as proteases, metabolic inhibitors and detergents may markedly reduce the mitotic activity of cultured lymphocytes. Among protease inhibitors phenyl methyl sulfonyl fluoride (PMFS) had no effect on phytohemagglutinin (PHA) induced stimulation while iodoacetamide (IAA) at a concentration of 0.01% significantly decreased PHA activation of lymphocytes. EDTA was inhibitory at a concentration of 2 x 10(-4) M. The effect of different non-ionic detergents in the PHA responsiveness was also evaluated. Tween 20 and Tween 80 were the least toxic. These findings for AChR may be relevant to preparation of other integral membrane proteins for assay of cellular immunity.