TITLE: Glucose-Stimulated Glucagon Secretion using Biorep Perifusion Machine - Mouse Islets
AUTHORS: Aliya Spigelman, Jocelyn Manning Fox, Patrick Macdonald

[DESCRIPTION]
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[STEPS]
?. [Day before experiment]
Isolate mouse islets as described in Mouse Islet Isolation protocol.
?. [Day before experiment]
Pick the isolated mouse islets into Mouse Islet Culture Media (>90% islet purity).   Mouse Islet Culture Media500 mL RPMI (11.1mMglucose) Gibco 1875-11950 mL FBS Canadian Origin Gibco12483-0205 mL Penicllin-StreptomycinGibco15140-122
500 mL RPMI (11.1mMglucose) Gibco 1875-11950 mL FBS Canadian Origin Gibco12483-0205 mL Penicllin-StreptomycinGibco15140-122
?. [Day before experiment]
Culture islets, up to 250 islets in 2 mL in a 35mm culture dish,  overnight in incubator at 37°C, 5% CO2.
?. [Solution preparation to be made fresh]
KRBH solution to be made fresh:mM Finalper 100mL total volumeNaCl1407 mL (2M stock)KCl3.6360 µL (1M stock)CaCl22.6260 µL (1M stock)NaH2PO40.550 µL (1M stock)MgSO40.550 µL (1M stock)HEPES5500 µL (1M stock)NaHCO320.0168 gEssentially fatty acid free BSA0.5mg/ml50 mgWarm above solution to 37°C (approximately 30min to1 hour). After warming the solution, pH to 7.45 with NaOH.
mM Finalper 100mL total volumeNaCl1407 mL (2M stock)KCl3.6360 µL (1M stock)CaCl22.6260 µL (1M stock)NaH2PO40.550 µL (1M stock)MgSO40.550 µL (1M stock)HEPES5500 µL (1M stock)NaHCO320.0168 gEssentially fatty acid free BSA0.5mg/ml50 mg
?. [Solution preparation to be made fresh]
Add glucose and/or additional treatments as required.
?. [Running the perifusion]
Set up the chambers and perifusion machine according to Biorep instructions.
?. [Running the perifusion]
Load islets into each chamber. The number of islets will vary by experiment and sensitivity of ELISA.
?. [Running the perifusion]
Run a 30 min pre-incubation as previously set and discard the perfusate.
?. [Running the perifusion]
Continue to run the perifusion protocol as samples are collected into a clean deep well plate(s).
?. [Running the perifusion]
Upon perifusion protocol is completion, 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.
?. [Running the perifusion]
Perfusate and islet content samples are stored at -20°C until ELISA
?. [Glucagon ELISA]
Perfusate is thawed and assayed using MSD glucagon Elisa kits. Vortex content sample and dilute 1:200
?. [Insulin Elisa]
If desired, samples are assayed for Insulin using Alpco Stellux mouse insulin ELISA. Vortex islet content and dilute 1:1000.
?. [Solutions preparation that can be made in advance]
Acid Ethanol for islet content collection95% ethanol150 mLAcetic Acid47 mLConcentrated HCl3 mL
95% ethanol150 mLAcetic Acid47 mLConcentrated HCl3 mL
?. [Running the perifusion]
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 preincubation step that should fill one 96 well plate. The perfusate from this step will be disgarded.
?. [Running the perifusion]
Prime the perifusion machine.