TITLE: Static glucagon secretion analysis of isolated islets
AUTHORS: Julien  Ghislain, Vincent  Poitout, Caroline  CT Tremblay, Marine Croze

[DESCRIPTION]
This protocol describes the steps to measure glucagon secretion in a static, 1-hour assay from isolated pancreatic islets. It is suitable for islets isolated from both rodents and humans. We routinely apply this protocol to assess alpha-cell function in response to low glucose concentrations and L-arginine but can be easily adapted to interrogate the response to a variety of secretagogues (eg. fatty acids, hormones). Briefly batches of 10 islets are pre-incubated in triplicate in KRB solution at 5.5 mM glucose twice for 20 min followed by incubation in 1 mM glucose with or without 10 mM L-arginine for 1 hour. Secreted glucagon is measured in the supernatant and intracellular glucagon content, after acid-alcohol extraction, by radioimmunoassay. This protocol is also suitable for assessing SST secretion, however we recommend increasing the islet number per well from 10 to at least 20 due to the relative lower levels of SST compared to glucagon.

[STEPS]
SECTION: Preparation of KRB solution and plates
1. Prepare KRB stocks, glucose and L-arginine solutions
KRB stock I: weigh 27.7 g NaCl bring to 1 L with milliQ water
KRB stock II: weigh 1.494 g CaCl2•2H2O and bring to 1 L with milliQ water
KRB stock III: weigh 0.648 g KH2PO4 and bring to 1 L with milliQ water
KRB stock IV: weigh 1.415 g KCl, 1.17 g MgSO4•7H2O and 8.52 g NaHCO3 and bring to 1 L with milliQ water
1 Molarity (M) Glucose: weigh 18.02 g and bring to 100 mL with milliQ water. Filter sterilize using a 0.2 μm filter.
SECTION: Preparation of KRB solution and plates
2. Prepare KRB solution
Determine the number of static conditions for the assay in order to prepare a sufficient volume of KRB. Remember that you will have two pre-incubation steps and the picking, along with extra media to wash between these steps. In a beaker combine equal volumes of the four KRB stock solutions to achieve the desired volume. Add 2.38 mg/mLHEPES powder and swirl to dissolve. Then add1 mg/mL BSA (fatty acid free), but do not mix as the BSA will stick to the sides. Cover with plastic wrap (put holes in top) and place in the 37 °C incubator for >60 min. Adjust the solution topH 7.35 using 1 Molarity (M) NaOH. The solution should start at ~ pH 7.2.
SECTION: Preparation of KRB solution and plates
3. 5.5 mM Glucose condition and islet picking and washing
Calculate the required volume of 5.5 millimolar (mM) Glucose in KRB for the two islet pre-incubations (1 mL/well) plus 1 mL/well and about 70 mL for the islet picking and wash steps. Add 5.5 µL of 1 Molarity (M) Glucose/ml of KRB. 

1 mM Glucose condition
Calculate the required volume of 1 millimolar (mM) Glucose in KRB for the static (1 mL/well) and add 1 µL of 1 Molarity (M) Glucose/ml of KRB needed.

1 mM Glucose plus 10 mM L-arginine condition
Calculate the required volume of 1 millimolar (mM) Glucose and 10 millimolar (mM) L-arginine in KRB for the static (1 mL/well) and add 1 µL of 1 Molarity (M) Glucose/ml of KRB . Then add L-arginine in powder form (1.75 mg/mL) and adjust to pH 7.35 with HCl.
 

SECTION: Preparation of KRB solution and plates
4. Prepare plates
Prepare islet picking plates by adding 1 mL of 5.5 millimolar (mM) Glucose in KRB to three wells of a 24-well plate for each static sample. 
Prepare pre-incubation plates by adding 1 mL of 5.5 millimolar (mM) Glucose in KRB to three wells of a 24-well plate for each static sample. Repeat this step for a second pre-incubation plate. Place these plates in an incubator with 5 % volume CO2 at 37 °C.
Prepare static incubation plate by adding 1 mL of experimental KRB (1 millimolar (mM) glucose alone or 1 millimolar (mM) glucose plus10 millimolar (mM) L-arginine) to three wells of a 24-well plate for each static sample. Place these plates in the incubator with 5 % volume CO2 at 37 °C.
SECTION: Islet Picking and Incubations
5. Following isolation, the islets should be allowed to recover in recovery medium (RPMI / 10% serum / 11.1 millimolar (mM) glucose) for 60 min at 37 °C. Wash the islets in a petri dish containing 20 mLof 5.5 millimolar (mM) Glucose in KRB.
Pick the islets (in triplicate batches of 10) into the islet picking plate wells.
Using a pipette transfer the islets from the picking plate to the first pre-incubation plate. Incubate at 37 °C  for 20 min.
Then transfer the islets to the second pre-incubation plate. Incubate at 37 °C  for 20 min.
Then transfer the islets to the incubation plate and incubate at 37 °C  for 60 min.
During the incubation, label two 1.5 mL tubes per sample for collection of the KRB media containing secreted glucagon. Prepare and label 1.5 mL tubes filled with 1 mLacidified ethanol (75 % (v/v) ethanol / 1.5 % (v/v) HCl) for glucagon content analysis.
At the end of the static incubation, collect the islets and transfer them to the tubes pre-filled with acidified ethanol. Cap the vials and store at -20 °C overnight.
Then, transfer the media from each well of the static incubation into a 1.5 mL tube, centrifuge at 4000 rpm, 5 min, 4 °C , transfer the supernatant to a new 1.5 mLtube and store at -20 °C until ready to complete the glucagon assay.
The next day retrieve the glucagon content analysis tubes, vortex and centrifuge at 4000 rpm, 5 min, 4 °C . Transfer the supernatant to labelled 1.5 mLtubes. Store the glucagon content samples at -20 °C, until ready to complete the glucagon assay.
SECTION: Radioimmunoassay
6. Radioimmunoassay kits are used to measure glucagon levels. Several kits are available from MilliporeSigma. For the protocol please refer to the manufacturer's instruction.