A microtiter plate assay using cellulose acetate filters for measuring cellular [3H]serotonin release

Abstract

An assay system for measuring the release of tritium-labeled serotonin that accompanies cellular degranulation is described. The assay is carried out with [3H]serotonin loaded rat basophilic leukemia (RBL) cells that have been sensitized with IgE, and aliquots of these are combined with dilutions of specific antigen in the wells of a microtiter plate together with appropriate control samples. The released activity in all of the wells is harvested simultaneously and quickly using commercial cellulose acetate filters which are counted following addition of scintillation fluor. In many applications this plate assay is faster and more convenient than the conventional test tube assay, and the results obtained are shown to be comparable. For the plate assay, the effect of cell concentration and some other conditions are shown. In general it is found that greatly improved results can be obtained if the cells are cultured overnight during the [3H]serotonin incorporation. With this assay it is shown that RBL cells also release incorporated 51Cr specifically during degranulation.