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

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

A	B
500mL DMEM (5mM glucose)	Gibco 11885
50 mL FBS Canadian Origin	Gibco 12483-020
5 mL Penicillin- Streptomycin	Gibco 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

A	B
500 mL RPMI (11.1mMglucose)	Gibco 1875-119
50 mL FBS Canadian Origin	Gibco12483-020
5 mL Penicillin-Streptomycin	Gibco15140-122

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.

Acid Ethanol for islet content collection

A	B
95% ethanol	150 mL
Acetic Acid	47 mL
Concentrated HCl	3 mL

KRBH solution to be made fresh on day of perifusion:

A	B	C
	mM Final	per 100mL total volume
NaCl	115	5.75 mL (2M stock)
KCl	5	500 µL (1M stock)
CaCl2	2.5	250 µL (1M stock)
MgCl2	1	100 µL (1M stock)
HEPES	10	1 mL (1M stock)
NaHCO3	24	0.2g
BSA	0.1% w/v	0.1g

Mix chemicals listed in the above table in milliQ water (approximately 80mL ). Warm KRBH solution to 37°C (approximately 30min to1 hour). Once solution is warm, pH to 7.4 with NaOH and bring volume to 100mL . KRBH should be kept at 37°C for the duration of the experiment.

Add glucose and/or additional compounds as required.

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 -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 -20°C until 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.