Glucose-Stimulated Insulin Assay.

This protocol can be performed on cell lines (6 well plate, ~80% confluent with the same number of cells plated in each well) or islets (20 islets per well).

KRBH solution

mM

NaCl 115

KCl 5

NaHCO3 24

CaCl2 2.5

MgCl2 1

HEPES 10

BSA 2% w/v

pH 7.4

Stock solutions can be made and used (NaHCO3 should be made fresh on day of use)

For 100ml KRBH

ml

2M NaCl 5.75

1M KCl 0.5

1M NaHCO3 2.4

CaCl2 0.25

MgCl2 0.1

1M HEPES 1

2g BSA

pH 7.4

Glucose should then be added to the required concentrations. Usually low glucose = 2.8mM and high glucose = 11.1 or 16.7 mM. Drugs/compounds of interest can also be added to low and/or high glucose solutions as desired.

GSIS protocol

1. Preincubation: 2.8mM glucose solution, 1ml per well for 30min at 37degrees

2. Remove and discard solution.

3. Low glucose: 2.8mM glucose solution, 1ml per well for 1hr at 37degrees

4. Remove and save solution. Store at –20 until assay.

5. High glucose: 11.1mM glucose solution, 1ml per well for 1hr at 37degrees

6. Remove and save solution. Store at –20 until assay.

7. Islet/cell DNA or protein extraction and quantification can be performed if required.