title: Intracellular insulin quantification by cell-ELISA.
abstract: 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.
authors.affiliation: Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC, Canada J1K 2R1. Electronic address: prk.andersen@gmail.com.
authors.forename: Parker Lyng
authors.initials: PL
authors.lastname: Andersen
authors.affiliation: Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC, Canada J1K 2R1. Electronic address: Patrick.Vermette@USherbrooke.ca.
authors.forename: Patrick
authors.initials: P
authors.lastname: Vermette
mesh_terms: D000818:Animals; D002460:Cell Line; D002908:Chronic Disease; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006943:Hyperglycemia / Q000473:pathology; D007328:Insulin / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology; D051379:Mice; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D007328:Insulin
keywords: Cell number; Cell-ELISA; INS-1 cells; Intracellular insulin; MafA downregulation; Pancreatic islet culture and β-cells