Glucagon measurement from islet populations using a TR-FRET assay

Polydimethylsiloxane (PDMS) prepolymer (Sylgard 184) -Dow Corning (Midland, MI)

Chip has been conditioned with BSS for at least 30 minutes

Islets are washed by transferring them to a petri dish with pre-warmed BSS with desired concentration of glucose (we used 20 mM) and let sit in BSS for a few minutes (~2 min)

TR-FRET glucagon assay -Cisbio (Walthan, MA)

Dextrose -Fisher Scientific (Pittsburg, PA)

Polydimethylsiloxane (PDMS) prepolymer (Sylgard 184) -Dow Corning (Midland, MI)

All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless noted otherwise. All solutions were made with ultrapure DI water (NANOpure Diamond System, Barnstead International, Dubuque, IA). A balanced salt solution (BSS) was used for islet experiments which contained 125 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 2.4 mM CaCl2, 25 mM tricine, and brought to pH 7.4 before addition of 0.1% BSA.

HTRF 96 well low volume plates

Spectramax iD5 plate reader

PDMS device was fabricated using previously described methods¹

10,000** pg/ml stock glucagon was diluted following manufacturer's protocol for a serial dilution.

Final concentration was 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.6 and 0 pg/mL

The table below shows how a typical well plate was arranged for incubation.

Rows A,B and C are shown in blue and are for the calibration to be run in triplicate. Values are given for pg/mL.

The yellow and orange portion are marked for the fractions from the first and second islet experiment and the numbers correspond to the time stamp of the fraction. For example, the "0" corresponds to the perfusate from 0-2 min.

Citation

Open wells (- - -) can be used for the lysate dilutions described below

Islets were incubated in PIM(S) media (Prodo Labs).

Chip had been conditioned with BSS for at least 30 minutes.

Islets washed by transferring them to a petri dish with prewarmed BSS with desired concentration of glucose (we used 20 mM) and let sit in BSS for a few minutes (~2 min)

Islets were then transferred to the device in a 10 µL pipet and were positioned so that they could sediment into the islet chamber on the device

Once islets are loaded the top of the chip is dried and chamber is sealed with PCR tape

Inlet and outlet tubing are connected to the device

Flow from syringe pumps is started and run for ~10-15 minutes or at least until the volume of the chip and outlet tubing has been displaced twice. We used a flow rate of 5 uL/min. *Note- times will change depending on flow rate.

Collect fractions every 2 min into low volume Eppendorf tubes and seal once fraction is collected.

Remove islets from device with a 200 µL pipet by vacuum and transfer to a small centrifuge tube

Remove islets with a 10 µL pipet after centrifugation

Lysis is performed using an acid ethanol mixture as described previously²

Transfer lysate to a low adsorption vial

Dilute lysate appropriately ( we used 10:1 and 20:1) with BSS.

10 uL of TR-FRET reagents were added to each well, covered, and incubated overnight for 12-14 hours at room temperature in the dark.

The Spectramax ID5 plate reader was used to measure the TR-FRET signal according to the manufacturer's protocol.

Excitation light (360 nm) was set for 50 us, followed by a 100 us delay, and a 600 us recording time of the two FRET channels (620 and 665 nm). This timing protocol was repeated every 2 ms for 100 cycles.

FRET ratios are presented as the ratio of the emission at 665 nm to that at 620 nm multiplied by 10,000

The average FRET signal from the 0 pg mL1 standard solution was subtracted from all FRET measurements

Calibration curves were generated by averaging the FRET ratio from the three replicates of each standard glucagon solution and plotting these values vs. the concentration of glucagon

Using calibration convert FRET ratio to pg/mL glucagon for all fractions and diluted lysate samples

Use lysate dilution that fell within the calibration range to quantify the amount of glucagon in the lysate

To report data as a percent of glucagon secreted, the amount of glucagon from each fraction was summed and added to the total glucagon in the lysate.³

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