Updated protocols for measurement of GSIS and calcium imaging of islet clusters

Updated static GSIS protocol:

1. Preparation

1.1 Warm 3.3G KRB buffer, 16.7G KRB buffer and 30mM KCl KRB buffer (containing 3.3G) in 37°C water bath.

1.2 Pre-fill each assay chamber with 100ul 3.3G KRB buffer (put in incubator until use) and label collection tubes.

2. Equilibration

2.1 Hand pick sufficient number of islets and wash once with 3.3G KRB buffer.

2.2 Use 10ul pipette tip to transfer islets and incubate them in 2ml 3.3G KRB buffer in tissue non-treated 6-well for 1hr at 37°C, 5% CO2 incubator for equilibration.

3. Assay procedure

3.1 After equilibration, hand pick 5 islets of similar sizes (use 10ul pipette tip to transfer islets) and transfer them into the center of assay chamber pre-filled with 100ul 3.3G KRB buffer and incubate for 30min at 37°C, 5% CO2 incubator.

3.2 Collect all the 100ul supernatant and store at −30°C until measurement of the basal insulin secretion.

3.3 Immediately add 100ul 16.7G KRB buffer into each assay chamber and incubate for 30min at 37°C, 5% CO2 incubator.

3.4 Collect all the 100ul supernatant and store at −30°C until measurement of the glucose-stimulated insulin secretion.

3.5 Immediately add 100ul 30mM KCl KRB buffer (containing 3.3G) into each assay chamber and incubate for 30min at 37°C, 5% CO2 incubator.

3.6 Collect all the 100ul supernatant and store at −30°C until measurement of the KCl-stimulated insulin secretion.

3.7 Immediately add 100ul islet lysis buffer (acid-ethanol) into each chamber and transfer the lysis buffer containing all the 5 islets into EP tube. Manually break islet clusters and extract hormone for at least 24h. 

3.8 Add equal volume of 100ul 1M Tris-HCl (pH 7.5) buffer for neutralization. Store the neutralized lysates at −30°C until measurement. 

3.9 Human insulin is measured using ALPCO Human Insulin ELISA kit (80-INSHU-E01.1).

Updated calcium imaging protocol:

1. Islet cluster attachment

1.1 Collect primary islets or hPSC-derived islet clusters.

1.2 Islet clusters are attached to confocal imaging chamber pre-coated with Cell-Tak for overnight.

2. Calcium dye loading

2.1 For ex vivo calcium imaging, islet clusters are washed twice with KRB buffer briefly, incubated with 2 μM Cal-520 AM for 60 min in an incubator (37°C, 5% CO2). 

2.2 Wash twice with KRB buffer, and incubated further in an incubator (37°C, 5% CO2) for another 15 min without the dye. 

3. Islet cluster imaging

3.1 Stained islets are staged on spinning-disc confocal microscope. 

3.2 Time-lapse images are captured very 1s with 100ms exposure under 20× magnification. Capture time-lapse images under low glucose for 15min and under high glucose for 15min.

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