A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macrophages

A simple, objective, and semiautomated densitometric method for the quantitation of nitroblue tetrazolium (NBT) reduction by macrophages (MP) and other cells is described. Guinea pig peritoneal MP were cultured in monolayers in 96-well flat bottom tissue culture plates and covered by 100 μl per well of a 1 mg/ml solution of NBT with or without a stimulant of the oxidative burst. The plates were incubated at 37 C for various times (mostly for 1 hr), and the absorbance of the formazan deposits in the cells was measured with the aid of an automatic eight channel photometer fitted with a 550-nm filter. This type of instrument, originaly designed for the automatic reading of enzyme immunoassays performed in microtitration plates, reads and records in print-out form the absorbance of individual wells in the plate against a selected blank. Wells containing MP pretreated with iodoacetamide served as blanks; these cells were incapable of NBT reduction in the presence or absence of stimulants. This technique offers the following advantages: a.) it is objective and fast, and offers results in print-out form, b.) it measures absolute amounts of reduced NBT and not merely the proportion of NBT-reducing cells, c.) in comparison to other quantitative methods based on solvent extraction of reduced NBT, this technique is simpler requires less cells, and permits repeated measurements of the same samples, and d.) the test is performed directly in the plates being used for a variety of tissue culture techniques. By using this technique, we demonstrated the ability of oil-elicited guinea pig MP to reduce NBT in response to phorbol myristate acetate, the ionophore A23187, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and to concanavain A. The NBT reduction elicited by these stimulants did not parallel the results of cytochrome c reduction performed at the same time. For 2 stimulants, A23187 and FMLP, NBT reduction was a considerably more sensitive assay than was cytochrome c reduction.