Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay

A short term microcytotoxicity assay is described for studying the activation of MØ by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. ⁵¹Cr-labeled humor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of MØ, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of MØ activation by exogenous stimuli, since MØ that were not pretreated with activating agents did not exhibit cytotoxicity.