TITLE: Static Glucose-stimulated Insulin Secretion (GSIS) Protocol: Mouse Islets
AUTHORS: Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Static glucose stimulated insulin secretion (GSIS) protocol for mouse islets. </div></div>

[STEPS]
?. [Experimental Protocol]
Pick islets into 35mm non-tissue cultured coated (NTCC) dish and ‘wash’ islets with  of KRBH with low glucose (2.8mM).
2 mL
?. [Experimental Protocol]
Pick islets into new 35mm NTCC dish  in of low glucose KRBH and pre-incubate in incubator at  5 % CO2 for .
2 mL
37 °C
?. [Experimental Protocol]
Transfer islets into a new 35mm NTCC dish and add  of low glucose (2.8mM) KRBH and pre-incubate for at , 5 % CO2.
2 mL
37 °C
?. [Experimental Protocol]
Pick 15 islets into eppendorf tubes. Each treatment group should be done in triplicate.Control group is typically 2.8mM glucose for low glucose and  16.7mM glucose for high glucoseNumber of islets can be increased/decreased depending on ELISA kit sensitivity
?. [Experimental Protocol]
Gently add  of low glucose KRBH to the islets, and incubate for  at , 5 % CO2. Leave tube lids open.
500 µl
37 °C
?. [Experimental Protocol]
Close lids, gently invert tubes, and centrifuge at   to pellet islets.
Centrifuge: 1000 33, 1 min
?. [Experimental Protocol]
Collect  as much of the  supernatant as possible without disturbing the pellet. Store supernatant at  until insulin assay.
500 µl
-20 °C
?. [Experimental Protocol]
Gently add  of high glucose (16.7mM) KRBH to islets, and incubate for  with conditions at , 5 % CO2. Leave lids open.
500 µl
37 °C
?. [Experimental Protocol]
Close lids, gently invert tubes, and centrifuge at   to pellet islets.
Centrifuge: 1000 33, 1 min
?. [Experimental Protocol]
Collect  as much of the  supernatant as possible without disturbing the pellet. Store supernatant at  until insulin assay.
500 µl
-20 °C
-20 °C
?. [Experimental Protocol]
Add  of acid ethanol to the islets. Store tube at  until insulin assay.
500 µl
-20 °C
?. [Solution Preparation]
Prepare solutions as follows:  ABC1&nbsp;
mM Final
per 100mL total
2NaCl115
5.75mL (2M)
3KCl5
0.5mL (1M)
4NaHCO324
0.2g
5CaCl22.5
0.25mL (1M)
6MgCl21
0.1mL (1M)
7HEPES10
1mL (1M)
8BSA0.1% w/v
0.1g
Mix chemicals from above table in milliQ water (approximately ). Warm KRBH solution to  in incubator 37°C, 5% CO2 (Approximately 30 min).  Once solution is warmed, pH KRBH solution to  with NaOH and bring to volume (). KRBH Solution should be kept in incubator throughout the experiment.Add glucose and/or additional treatments as required.  ABC1per 50mL total
from 1M stock
from powder
22.8mM140µL
0.025g
316.7mM835µL
0.150g
Acid Ethanol:   AB195% Ethanol150mL
2Acetic Acid47mL
3Concentrated HCL3mL
ABC1&nbsp;
mM Final
per 100mL total
2NaCl115
5.75mL (2M)
3KCl5
0.5mL (1M)
4NaHCO324
0.2g
5CaCl22.5
0.25mL (1M)
6MgCl21
0.1mL (1M)
7HEPES10
1mL (1M)
8BSA0.1% w/v
0.1g
80 mL
37 °C
100 mL
ABC1per 50mL total
from 1M stock
from powder
22.8mM140µL
0.025g
316.7mM835µL
0.150g
AB195% Ethanol150mL
2Acetic Acid47mL
3Concentrated HCL3mL
?. [Day Before Secretion]
Pick mouse islets into Mouse Islet Culture Media until as close as possible to 100% purity.Mouse Islet Culture Media  AB1500ml RPMI 1640 (11.1mM glucose)
Gibco 11875-119
250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep
Gibco 15140-122
AB1500ml RPMI 1640 (11.1mM glucose)
Gibco 11875-119
250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep
Gibco 15140-122
?. [Day Before Secretion]
Culture islets overnight in incubator at 37°C, 5% CO2.
?. [ELISA]
Samples are assayed using ALPCO Stellux Rodent Insulin ELISA kit (Cat # 80-INSMR-CH10).Content samples are diluted with zero buffer 1:400.
?. [Day Before Secretion]
Mouse islets isolated as described in Mouse Islet Isolation protocol.