Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands

Cytometry. 1994 Aug 1;16(4):339-45. doi: 10.1002/cyto.990160408.

Abstract

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 (N alpha beta 1-biotinylinsulin (B-insulin) and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin (NBC-insulin)). The reference methods were 125I-insulin binding and the insulin-antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I-insulin binding and increase in fluorescence for B-insulin, NBC-insulin, and insulin-anti-insulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluorescence (79 +/- 9 channels) and the the insulin-antiinsulin antibody complexes the smallest (34 +/- 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 +/- 6 and 59 +/- 6 channels. The concentration reducing 125I-insulin binding by 50% was 1.1 x 10(-9) M for native insulin, 2.7 x 10(-9) M for B-insulin, 3.3 x 10(-9) M for NBC-insulin, and 6.6 x 10(-9) M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for 10(-6) 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.

MeSH terms

  • Animals
  • Biotin / analogs & derivatives*
  • Biotin / metabolism
  • Cattle
  • Flow Cytometry
  • Fluorescein-5-isothiocyanate / analogs & derivatives
  • Humans
  • Insulin / analogs & derivatives*
  • Insulin / metabolism*
  • Ligands
  • Lymphocytes / metabolism*
  • Multiple Myeloma / pathology
  • Protein Binding
  • Receptor, Insulin / metabolism*
  • Swine
  • Tumor Cells, Cultured

Substances

  • B-insulin
  • B29-biotin insulin
  • Insulin
  • Ligands
  • NBC-insulin
  • insulin, fluorescein-isothiocyanated-
  • Biotin
  • Receptor, Insulin
  • Fluorescein-5-isothiocyanate