The binding of insulin to cultured IM-9 human lymphocytes was studied by flow cytometry using FITC-insulin and biotinylated insulins coupled to streptavidin-phycoerythrin (NaB'-biotinylinsulin (B-insulin) and NaB1 -( biotinyl-Eaminocaproy1)insulin (NBC-insulin)). The reference methods were 1251-insulin binding and the insulin-antiinsulin antibody complexes for flow cytometry. There was a close correlation between 1251-insulin binding and increase in fluorescence for B-insulin, NBC-insulin, and insulin-antiinsulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluorescence (79 f 9 channels) and the the insulin-antiinsulin antibody complexes the smallest (34 2 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 f 6 and 59 f 6 channels. The concentration reducing 1251-insulin binding by 50% was 1.1 x lo-' M for native insulin, 2.7 x lo-' M for B-insulin, 3.3 x lo-' M for NBCinsulin, and 6.6 x lo-' M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for M FITC-insulin. These results suggest that B-insulin and NBC-insulin are suitable ligands for insulin binding studies using flow cytometry. This two-step procedure is easier than the insulin-antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC-insulin less suitable. o

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