Static insulin secretion analysis of isolated islets
Caroline CT Tremblay, Julien Ghislain, Vincent Poitout, Marine Croze
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.
Steps
Preparation of KRB solution and plates
Prepare KRB stocks KRB stock I: weigh 27.7g NaCl bring to 1L with milliQ waterKRB stock II: weigh 1.494g CaCl2•2H2O and bring to 1L with milliQ waterKRB stock III: weigh 0.648g KH2PO4 and bring to 1L with milliQ waterKRB stock IV: weigh 1.415g KCl, 1.17g MgSO4•7H2O and 8.52g NaHCO3and bring to 1L with milliQ water1Molarity (M) Glucose: weigh 18.02g and bring to 100mL 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 2.38mg/mLHEPES powder and swirl to dissolve. Then add1mg/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 >1h 0m 0s. Adjust the solution to7.35 using 1Molarity (M) NaOH. The solution should start at ~ 7.2.
2.8 mM Glucose condition and islet picking and washing Calculate the required volume of 2.8millimolar (mM) Glucose in KRB for the two islet pre-incubations and static (1mL/well) plus 1mL/well and about 70mL for the islet picking and wash steps. Add 2.8µL of 1Molarity (M) Glucose/ml of KRB. 16.7 mM Glucose condition Calculate the required volume of 16.7millimolar (mM) Glucose in KRB for the static (1mL/well) and add 16.7µL of 1Molarity (M) Glucose/ml of KRB needed.
Prepare plates Prepare islet picking plates by adding 1mL of 2.8millimolar (mM) Glucose in KRB to three wells of a 24-well plate for each static sample. Prepare pre-incubation plates by adding 1mL of 2.8millimolar (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 1mL 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
Following isolation, the islets should be allowed to recover in recovery medium (RPMI / 10% (v/v) serum / 11.1millimolar (mM) glucose) for 1h 0m 0s at 37°C. Wash the islets in a petri dish containing 20mLof 2.8millimolar (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 0h 20m 0s.Then transfer the islets to the second pre-incubation plate. Incubate at 37°C for 0h 20m 0s.Then transfer the islets to the incubation plate and incubate at 37°C for 1h 0m 0s.During the incubation, label two 1.5mL tubes per sample for collection of the KRB media containing secreted insulin. Prepare and label 1.5mL tubes filled with 1mLacidified 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 1.5mL tube, centrifuge at 4000rpm,4°C , transfer the supernatant to a new 1.5mLtube 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 4000rpm,4°C . Transfer the supernatant to labelled 1.5mLtubes. Store the insulin content samples at -20°C, until ready to complete the insulin assay.
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.
