Intracellular insulin quantification by cell-ELISA

• Relative intracellular insulin content in cultured cells was resolved with cell-ELISA.
• Relative cell density was resolved with Janus Green B staining.
• Relative insulin content was confirmed by fluorescence visualization.
• Insulin content was normalized to cell density by assaying identical cultures.
• The insulin cell-ELISA assay is applicable to INS-1 cells and primary islet cultures.

Current methods of monitoring insulin in culture are limited to soluble insulin (secretions or lysates) or synthetic gene reporter analyses. We present an insulin-specific cell enzyme-linked immunosorbent assay (cell-ELISA) to assess relative intracellular insulin protein content of adherent cultures, normalized to cell density with further immunocytochemical verification of insulin-expressing cells within identical cultures. The protocol was optimized and validated using an insulin-expressing cell line (INS-1) by confirming direct relations between intracellular insulin content and insulin-expressing cell density, in a glucose exposure-dependent manner. Utility was demonstrated by identifying multiple INS-1 clones lowly expressing insulin following lentiviral particle delivery of interference RNA intended to down-regulate one of the insulin gene-directed transcription factors, MafA. The cell-ELISA was also applied to monitor insulin content within partially dissociated primary mouse islet cultures. This technique allows for the first time routine analysis of intracellular insulin protein in vitro suitable for investigating islet cell biology by means of multiple parameter screening.