Activated rat neutrophils. A sequential quantitative assay for aggregation and NBT reduction

We present here a rapid, sensitive, and convenient approach for the analysis of activated Lewis rat PMNs based on detecting separately, or in tandem, PMN aggregation and PMN reduction of nitroblue tetrazolium (NBT). These responses are quantitated using an ELISA scanner which can rapidly measure optical densities of cell cultures in microtiter plates. Aggregation induced by as little as 0.005 micrograms/ml of phorbol myristate acetate (PMA), 0.01 micrograms/ml lipopolysaccharide (LPS), or a 1:160 dilution of lymphokine-containing rat serum can be detected employing this approach. NBT reduction was induced by as little as 0.01 micrograms/ml PMA. Blocking studies employing 2-deoxyglucose, iodoacetamide, and polymyxin B gave the expected results and confirmed that these assays detect cellular responses to soluble stimuli. Using this technology the effects of PMA and LPS on rat peritoneal exudate PMNs were evaluated. Rat PMNs appeared less sensitive to LPS than human PMNs and also reduced NBT more slowly following stimulation with PMA. Because of the slowness in NBT reduction following stimulation, NBT reduction can be evaluated, in tandem, after measuring aggregation. The simplicity of this system, coupled with the speed with which large numbers of microcultures can be read and the low number of cells required, make this approach for studying responses especially attractive.