A microplate format assay for histamine release by activated rat peritoneal mast cells with accompanying microplate format spectrofluorometric assay of histamine is demonstrated. Reproducability and linearity of the histamine assay are comparable to the historical large format assay. Transfer of samples in the 96 well format from histamine release through spectrofluorometric assay greatly expedites handling and provides for better independence of each replicate. Several new observations were made. The microplate format assay was used to investigate the kinetics of fluorescence development in the histamine assay before and after acidification, and the kinetics of assay at room temperature. A wide range dose response to compound 48/80 was done both as a sample experiment included to show a typical size experiment easily done in this format, and to internally control responses throughout too wide a dose range to normally be done in a single experiment. A reproducible depression of responses at higher than optimal doses of 48/80 was observed. These observations indicate the effectiveness and practicality of the microplate assay, improve histamine assay parameters, and raise new questions about histamine release.
|State||Published - 1 Dec 1997|