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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.

journal_name

Exp Cell Res

journal_title

Experimental cell research

authors

Andersen PL,Vermette P

doi

10.1016/j.yexcr.2016.06.014

subject

Has Abstract

pub_date

2016-09-10 00:00:00

pages

14-23

issue

1

eissn

0014-4827

issn

1090-2422

pii

S0014-4827(16)30164-1

journal_volume

347

pub_type

杂志文章

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