Jun 16, 2022
Static insulin secretion analysis of isolated islets
- Julien Ghislain1,
- Vincent Poitout2,
- Caroline CT Tremblay1,
- Marine Croze1
- 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada;
- 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada
Protocol Citation: Julien Ghislain, Vincent Poitout, Caroline CT Tremblay, Marine Croze 2022. Static insulin secretion analysis of isolated islets . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61dxkvqe/v1
Manuscript citation:
Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice. Croze ML, Flisher MF, Guillaume A, Tremblay C, Noguchi GM, Granziera S, Vivot K, Castillo VC, Campbell SA, Ghislain J, Huising MO, Poitout V. Mol Metab. 2021 Mar;45:101166. doi: 10.1016/j.molmet.2021.101166. PMID: 33484949
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 29, 2022
Last Modified: June 16, 2022
Protocol Integer ID: 60048
Keywords: pancreatic islet, somatostatin, insulin, glucose-stimulated insulin secretion
Funders Acknowledgement:
NIH
Grant ID: 5R01DK058096-15
CIHR
Grant ID: 0406014428
NSERC
Grant ID: 51047
Abstract
This protocol describes the steps to measure insulin 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 beta-cell function in response to glucose 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 2.8 mM glucose twice for 20 min followed by incubation in either 2.8 mM or 16.7 mM glucose for 1 hour. Secreted insulin is measured in the supernatant and intracellular insulin 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 insulin.
Materials
Sodium ChlorideFisher ScientificCatalog #S271
Calcium Chloride DihydrateFisher ScientificCatalog #C79 Potassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791
Potassium ChlorideSigma – AldrichCatalog #746436
Magnesium sulfate heptahydrate (MgSO4)Millipore SigmaCatalog #63138
Sodium bicarbonateSigma AldrichCatalog #S6014
HEPESSigma AldrichCatalog #H6147
D-( )-GlucoseSigma AldrichCatalog #G-7528
Fatty Acid Free Heat Shock Bovine Serum Albumin Powderequitech bio, inc.Catalog #BAH66-0500
Ethanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
RPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
FCS (Fetal Calf Serum) Life Technologies
Rat insulin RIASigma AldrichCatalog #RI-13K
Human Insulin-Specific RIASigma AldrichCatalog #HI-14K
Preparation of KRB solution and plates
Preparation of KRB solution and plates
Prepare KRB stocks
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.
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/mL HEPES 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 >01:00:00 . Adjust the solution to7.35 using 1 Molarity (M) NaOH. The solution should start at ~ 7.2 .
1h
2.8 mM Glucose condition and islet picking and washing
Calculate the required volume of 2.8 millimolar (mM) Glucose in KRB for the two islet pre-incubations and static (1 mL /well) plus 1 mL /well and about 70 mL for the islet picking and wash steps. Add 2.8 µL of 1 Molarity (M) Glucose/ml of KRB.
16.7 mM Glucose condition
Calculate the required volume of 16.7 millimolar (mM) Glucose in KRB for the static (1 mL /well) and add 16.7 µL of 1 Molarity (M) Glucose/ml of KRB needed.
Note
Often there are other reagents to be added to the final static conditions, such as fatty acids, inhibitors or agonists. These additional components may require that separate KRB solutions be prepared. In the case of fatty acids addition prepare the KRB solution without BSA.
Prepare plates
Prepare islet picking plates by adding 1 mL of 2.8 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 2.8 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 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 .
Islet Picking and Incubations
Islet Picking and Incubations
2h 40m
2h 40m
Following isolation, the islets should be allowed to recover in recovery medium (RPMI / 10 % (v/v) serum / 11.1 millimolar (mM) glucose) for 01:00:00 at 37 °C . Wash the islets in a petri dish containing 20 mL of 2.8 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 00:20:00 .
Then transfer the islets to the second pre-incubation plate. Incubate at 37 °C for 00:20:00 .
Then transfer the islets to the incubation plate and incubate at 37 °C for 01:00:00 .
During the incubation, label two 1.5 mL tubes per sample for collection of the KRB media containing secreted insulin. Prepare and label 1.5 mL tubes filled with 1 mL acidified ethanol (75 % (v/v) ethanol / 1.5 % (v/v) HCl) for insulin 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, 4°C, 00:05:00 , transfer the supernatant to a new 1.5 mL tube and store at -20 °C until ready to complete the insulin assay.
The next day retrieve the insulin content analysis tubes, vortex and centrifuge at 4000 rpm, 4°C, 00:05:00 . Transfer the supernatant to labelled 1.5 mL tubes. Store the insulin content samples at -20 °C , until ready to complete the insulin assay.
2h 50m
Radioimmunoassay
Radioimmunoassay
Radioimmunoassay kits are used to measure insulin levels. Several kits are available from MilliporeSigma. For the protocol please refer to the manufacturer's instruction.