TITLE: Static Incubation of Pancreatic Islets
AUTHORS: Islet  and Pancreas Analysis Core

[DESCRIPTION]
This SOP defines the methods used by the Vanderbilt Diabetes Center Islet and Pancreas Analysis (IPA) Core for static incubation of pancreatic islets isolated from mouse or human tissue. See also our islet isolation protocol.

[STEPS]
SECTION: Reagent preparation
1. RPMI with 5.6 mM glucose (cell culture media):

Remove 5 mL from a 500 mL bottle of RPMI, and add 5 mL penicillin/streptomycin. Close bottle and invert to mix.
Add 0.505 g glucose to RPMI, invert to mix, and incubate at room temperature for 60 min to allow glucose to go into solution.
In a BSL-2 culture hood, vacuum filter 450 mL RPMI.
Add 50 mL FBS.
 


SECTION: Reagent preparation
2. DMEM (static incubation media):

Add the following ingredients into a 1 L Erlenmeyer flask.
8.28 g DMEM
3.2 g sodium bicarbonate
0.58 g L-gutamine
1.11 g HEPES
0.11 g sodium pyruvate
1 g bovine serum albumin
70 mg ascorbate
1 L deionized water

Place a stir bar into the flask and situate on top of a magnetic stir plate. Stir at medium-high speed for 15 min to allow all ingredients to go into solution.
SECTION: Reagent preparation
2.1. 
Aliquot 500 mL of DMEM media into each of 2 plastic storage bottles to make 5.6 mM glucose and 16.7 mM glucose solutions.
SECTION: Reagent preparation
2.2. DMEM with 5.6 mM glucose:
Add 0.505 g glucose to 500 mL prepared DMEM media.
Incubate for 60 min to allow glucose to go into solution.
Vacuum-filter media.
SECTION: Reagent preparation
2.3. DMEM with 16.7 mM glucose:
Add 1.504 g glucose to 500 mL prepared DMEM media.
Incubate for 60 min to allow glucose to go into solution.
Vacuum-filter media.
SECTION: Reagent preparation
3. Acid ethanol (for hormone extraction):
Pipet 5.5 mL 95% ethanol into a 15 mL conical tube.
Add 50µL hydrochloric acid.
Invert to mix.
SECTION: Static incubation
4. Pipet 5 mL RPMI with 5.6 mM glucose into each of two 6-cm cell culture dishes.
SECTION: Static incubation
5. Using an electronic pipette and an inverted microscope, pass islets into the first plate, and then into the second.
SECTION: Static incubation
6. Place islets into a 37 °C cell culture incubator with 5% CO2 and leave overnight for a minimum of 1080 min.

SECTION: Static incubation
7. Pipet 5 mL of DMEM with 5.6 mM glucose into a 6-cm cell culture dish.
SECTION: Static incubation
9. Label a 12-well plate for static incubation according to your experimental design. In the example below, two conditions are shown, low glucose (5.6 mM) and high glucose (16.7 mM). These conditions can be repeated in subsequent rows and/or plates for biological replicates as desired.

  
SECTION: Static incubation
9.1. Pipet 2 mL of the appropriate media into each well. Note that each condition requires two wells, a "wash" well and an "incubation" well.
SECTION: Static incubation
10. Under microscope guidance, place around 20 islet equivalents (IEQs) into each wash well. This is typically a combination of 12 small, 5 medium, and 2 large islets. Use the chart below to measure islet sizes.


      
 

SECTION: Static incubation
11. Once islet aliquots have been distributed to wash wells, place the 12-well plate onto the stereomicroscope stage, swirl the plate to move islets to center of each well, and transfer the islets into the subsequent incubation wells. Use no more than 20 µL media for the transfer to avoid dilution of culture media.
SECTION: Static incubation
12. Cover the 12-well plate and place in 37 °C incubator with 5% CO2 for 70 min.

 

SECTION: Media collection and hormone extraction
13. Remove plate from incubator and place on ice to cool down for 10 min. During this time, label two 2-mL microcentrifuge tubes for each incubation well (islet aliquot): the first for islet extract and the second for media.
SECTION: Media collection and hormone extraction
19. Following the 1440 min incubation in step 17, spin down islet extract tubes for 5 min at 3000 rcf.
SECTION: Media collection and hormone extraction
21. Perform hormone assay of your choice.
SECTION: Static incubation
8. Using an electronic pipette and an inverted microscope, transfer islets from overnight incubation plate (step 6) into the 6-cm plate with DMEM media. Islets will be picked from this plate and placed into the 12-well static incubation plate described in steps 9-10.
SECTION: Media collection and hormone extraction
16. Remove supernatant from islet pellets, and add 200 µL acid ethanol to each tube.
SECTION: Media collection and hormone extraction
17. Transfer islet extract tubes to 4 °C to begin incubation for 1440 min. Meanwhile, proceed to step 18.
SECTION: Media collection and hormone extraction
18. After islets are removed, transfer 1.5 mL media from each incubation well into the second of each pair of labeled microcentrifuge tubes. Store at -20 °C.
SECTION: Media collection and hormone extraction
14. Return plate to the stereomicroscope stage, swirl the plate to move islets to center of each well, and pipet the islets in each incubation well into the first of each pair of labeled microcentrifuge tubes.
SECTION: Media collection and hormone extraction
15. Centrifuge the islet tubes for 3 min at 200 rcf.
SECTION: Media collection and hormone extraction
20. Transfer three 50-µL supernatant aliquots from each islet extract tube into prelabeled 2-mL screwcap tubes and store at -80 °C.