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Intracellular insulin quantification by cell-ELISA.

Parker Lyng Andersen1, Patrick Vermette1

  • 1Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boul. de l'Université, Sherbrooke, QC, Canada J1K 2R1.

Experimental Cell Research
|June 26, 2016
PubMed
Summary

Researchers developed a new cell-ELISA to measure intracellular insulin protein in cell cultures. This method aids in studying islet cell biology and insulin gene expression.

Keywords:
Cell numberCell-ELISAINS-1 cellsIntracellular insulinMafA downregulationPancreatic islet culture and β-cells

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Endocrinology

Background:

  • Current methods for monitoring insulin in cell cultures, such as analyzing soluble insulin or using gene reporters, have limitations.
  • Assessing intracellular insulin protein levels in adherent cell cultures is challenging.

Purpose of the Study:

  • To develop and validate an insulin-specific cell enzyme-linked immunosorbent assay (cell-ELISA) for quantifying intracellular insulin protein in adherent cell cultures.
  • To enable routine analysis of intracellular insulin protein in vitro for islet cell biology research.

Main Methods:

  • Developed an insulin-specific cell-ELISA for adherent cultures, normalized to cell density.
  • Validated the assay using INS-1 cells, demonstrating a glucose-dependent relationship between intracellular insulin and cell density.
  • Utilized immunocytochemistry for verification of insulin-expressing cells within the same cultures.

Main Results:

  • The cell-ELISA accurately reflects intracellular insulin content in relation to cell density.
  • The assay successfully identified INS-1 clones with reduced insulin expression after MafA knockdown via RNA interference.
  • Demonstrated utility in partially dissociated primary mouse islet cultures.

Conclusions:

  • The novel cell-ELISA provides a reliable method for routine analysis of intracellular insulin protein in vitro.
  • This technique facilitates multi-parameter screening for investigating islet cell biology and insulin regulation.