TITLE: Dynamic Glucose-Stimulated Insulin Secretion using Biorep Perifusion Machine (ver peri4.2) - Human or Mouse Islets
AUTHORS: Aliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald

[DESCRIPTION]
General protocol for Dynamic Glucose-Stimulated Insulin Secretion using Biorep Perifusion Machine - Human or Mouse Islets.

[STEPS]
SECTION: Day before experiment
1. 
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


 

SECTION: Day before experiment
2. 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

 


 
 
SECTION: Day before experiment
3. 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.
SECTION: Solutions preparation that can be made in advance
4. Acid Ethanol for islet content collection



 

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


 


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

 


SECTION: Solution preparation to be made fresh
6. Add glucose and/or additional compounds as required.
SECTION: Running the perifusion
7. Set up the chambers and perifusion machine according to Biorep instructions.



SECTION: Running the perifusion
8. 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.
SECTION: Running the perifusion
9. Prime the perifusion machine.
SECTION: Running the perifusion
10. Load islets into each chamber. The number of islets will vary by experiment and sensitivity of ELISA.
SECTION: Running the perifusion
11. Run a 30 min pre-incubation as previously set (step 1 of perifusion machine protocol) and discard the perfusate. 
SECTION: Running the perifusion
12. Continue to run the perifusion protocol, collecting samples into a clean deep well plate(s). 
SECTION: Running the perifusion
13. Upon completion of perifusion protocol, seal the place deep well plates at store at -20 °C until ELISA . 


SECTION: Running the perifusion
14. 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

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