May 31, 2023

Public workspaceDynamic Glucose-Stimulated Insulin Secretion using Biorep Perifusion Machine (ver peri4.2) - Human or Mouse Islets

  • 1University of Alberta
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Protocol CitationAliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald 2023. Dynamic Glucose-Stimulated Insulin Secretion using Biorep Perifusion Machine (ver peri4.2) - Human or Mouse Islets. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4kdqzvo5/v1
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: May 05, 2021
Last Modified: May 31, 2023
Protocol Integer ID: 49689
Abstract
General protocol for Dynamic Glucose-Stimulated Insulin Secretion using Biorep Perifusion Machine - Human or Mouse Islets.
Materials
Penicillin-Streptomycin- Gibco 15140122
RPMI 1640- Gibco 11875
DMEM- Gibco 11885
FBS (Canadian origin)- Gibco 12483-020

Sodium Chloride- Fisher Scientific BP358-212
Potassium Chloride- Sigma Aldrich P9541
Calcium Chloride- Sigma Aldrich C4901
Magnesium Chloride Hexahydrate- EMD millipore MX0045
HEPES- Fisher BP310-500
Sodium bicarbonate- Sigma Aldrich S5761
Bovine serum albumin- Sigma Aldrich A7906
D Glucose- Sigma Aldrich G8270

Biorep perifusion machine (ver peri4.2)
Deep well 96 well plates Fisher 12-565-606 or Greiner Bio-One 780280FD

Alpco stellux human Insulin ELISA: 80-INSHU-CH10
Alpco stellux Rodent Insulin ELISA: 80-INSMR-CH01


Day before experiment
Day before experiment

For human islets:

Pick human islets of similar size and shape into Human Islet Culture Media until as close as possible to 100% purity.

Human Islet Culture Media


AB
500mL DMEM (5mM glucose)Gibco 11885
50 mL FBS Canadian OriginGibco 12483-020
5 mL Penicillin- StreptomycinGibco 15140-122

For mouse islets:

Isolate mouse islets as described in Mouse Islet Isolation protocol.

Pick the isolated mouse islets into Mouse Islet Culture Media (>90% islet purity).

Mouse Islet Culture Media

AB
500 mL RPMI (11.1mMglucose) Gibco 1875-119
50 mL FBS Canadian Origin Gibco12483-020
5 mL Penicillin-StreptomycinGibco15140-122


For human and mouse islets:

Culture islets, up to 250 islets in 2 mL of media, in a 35mm non-treated culture dish, overnight in incubator at 37°C, 5% CO2.
Solutions preparation that can be made in advance
Solutions preparation that can be made in advance
Acid Ethanol for islet content collection



AB
95% ethanol150 mL
Acetic Acid47 mL
Concentrated HCl3 mL

Solution preparation to be made fresh
Solution preparation to be made fresh
KRBH solution to be made fresh on day of perifusion:


ABC
mM Finalper 100mL total volume
NaCl1155.75 mL (2M stock)
KCl5500 µL (1M stock)
CaCl22.5250 µL (1M stock)
MgCl21100 µL (1M stock)
HEPES101 mL (1M stock)
NaHCO3240.2g
BSA0.1% w/v0.1g


Mix chemicals listed in the above table in milliQ water (approximately Amount80 mL ). Warm KRBH solution to Temperature37 °C (approximately 30min to1 hour). Once solution is warm, pH to 7.4 with NaOH and bring volume to Amount100 mL . KRBH should be kept at Temperature37 °C for the duration of the experiment.



Add glucose and/or additional compounds as required.
Running the perifusion
Running the perifusion
Set up the chambers and perifusion machine according to Biorep instructions.



Set the protocol with glucose and experimental conditions (high KCl, inhibitors etc) and frequency of collection as needed using a flow rate of 100 μL/min for all steps.

Set step 1 of the perifusion protocol to total 30 min. The number of replicates will vary based on number of lanes and plate orientation. This is a pre-incubation step that should fill one 96 well plate. The perfusate from this step will be discarded.
Prime the perifusion machine.
Load islets into each chamber. The number of islets will vary by experiment and sensitivity of ELISA.
Run a 30 min pre-incubation as previously set (step 1 of perifusion machine protocol) and discard the perfusate.
Continue to run the perifusion protocol, collecting samples into a clean deep well plate(s).
Upon completion of perifusion protocol, seal the place deep well plates at store at Temperature-20 °C until ELISA .


To collect insulin content, run the chambers dry, so islets are on the filter paper but no liquid remains. Place the filter paper with islets into a 1.5 mL tube with 500 μL of acid ethanol. Store at Temperature-20 °C until ELISA

Insulin Elisa
Insulin Elisa
Samples are assayed for Insulin using Alpco Stellux human or mouse insulin ELISA. Vortex insulin content samples and dilute 1:400 prior to assay.