TITLE: Mouse Islet Perifusion (3-stimuli protocol)
AUTHORS: Islet and Pancreas Analysis Core

[DESCRIPTION]

Our perifusion setup consists of 4 independently-driven channels, which allows for up to 4 different groups of islets to be studied simultaneously. Each channel has an intake catheter, a peristaltic pump, a chamber to hold the islets, and a fraction collector.

This protocol details stimulation of mouse islets with 3 secretagogues (high glucose, IBMX, and KCl). The flow rate for the perifusion is 1 mL/minute, and fractions are collected every 3 minutes. The animation below illustrates the protocol.
  


[STEPS]
SECTION: General Perifusion Startup
1. Fill perifusion water bath with deionized water to about 1 inch from the top and set the temperature to 37 °C.
SECTION: Preparation of Base Perifusion Media and Secretagogues
4. Prepare base perifusion media by combining reagents below in a 1-L Erlenmeyer flask. Add 1 L of deionized water and mix on a magnetic stir plate for at least 15 min until dissolved.


3.2 g  
0.58 g  
0.11 g 
1.11 g   
8.28 g  
1 g  
70 mg  
 

SECTION: Setup of Secretagogues in Perifusion Water Bath
9. Place the bottles of media in the water bath to warm for at least 10 min before beginning the perifusion.
SECTION: Islet Preparation and Loading
14. Prepare a 6-cm dish with 5 mL islet picking media for each channel to be run. Place freshly-isolated mouse islets on the stage of inverted microscope and view the islets using the 4x objective. Using the reticle in the 10x eyepiece, size the islets according to the chart below.

  

Handpick islets using a 200-µL micropipette and transfer to the 6-cm tissue culture dish containing islet picking media. Record islet number and size to calculate IEQ.
 

SECTION: Collection of Perifusate Fractions
25. Collect 10 preliminary fractions using 5.6 mM glucose media to rinse the islets and to synchronize intake pump speed to deliver 1 mL per minute fractions, collecting 3 mL (3 minute) per fraction.
SECTION: Collection of Perifusate Fractions
27. Begin to collect fractions, changing media/secretagogues at predetermined timepoint based on fraction collector timer (see sample protocol below).When the fraction collector moves at the desired timepoint, pause the intake pump.

  
SECTION: Islet Recovery and System Cleanup
31. When perifusion is complete, stop intake and air uptakes pumps and close stopcocks to air uptake and media outlet lines.
SECTION: Islet Hormone Extraction
38. Using 6-cm dish with recovered islets, quantify IEQ ( ) and perform imaging ( ) to calculate IEQ recovered.
 

SECTION: Data Collection and Analysis
43. Open the darkfield image in the cellSens software. Using the manual HSV threshold function, segment the islet tissue channel.
SECTION: General Perifusion Startup
2. Label perifusion tubes with date, islet type, fraction number, and any other identifying information. Position the fraction collector trays for perifusion, and load the tubes into the fraction collector.
SECTION: General Perifusion Startup
3. Rinse the tubing with deionized water at max pump speed for 15 min, then place new frits into the islet chamber.
SECTION: Preparation of Base Perifusion Media and Secretagogues
5. Prepare 5.6 mM glucose media by adding 0.5549 g   to 550 mL base perifusion media in a 500-mL bottle and mix until dissolved.
SECTION: Preparation of Base Perifusion Media and Secretagogues
6. Prepare 16.7 mM high glucose media by adding 0.7522 g   to 250 mL base perifusion media in a 250-mL bottle and mix until dissolved.
SECTION: Preparation of Base Perifusion Media and Secretagogues
7. Prepare 5.6 mM glucose with 20 mM KCl by adding 0.149 g   to 100 mL 5.6 mM glucose media.

SECTION: Preparation of Base Perifusion Media and Secretagogues
8. Prepare 16.7 mM glucose with 100 µM IBMX by first weighing approximately 10 mg 
 in a microcentrifuge tube.
SECTION: Setup of Secretagogues in Perifusion Water Bath
10. Replace Pyrex orange caps with 4-Luer (+1) caps on every bottle of media to be used and tape over the holes so that gas cannot escape.
SECTION: Setup of Secretagogues in Perifusion Water Bath
11. Turn on gas (  O2,  % CO2) and insert one gas line to each bottle cap. Block additional holes in caps with tape. Make sure that gas line is suspended above the media and not submerged in the liquid.
SECTION: Setup of Secretagogues in Perifusion Water Bath
12. Place intake catheters into baseline media bottle, making sure that they rest on the bottom of the bottle. Run baseline media through the chambers at maximum pump speed for about 10 min while islets are being aliquoted for perifusion. Discard flow-through.

SECTION: Islet Preparation and Loading
16. Perform islet imaging:

Place the dish containing clean islets on the stage of a stereomicroscope equipped with a high-resolution camera and swirl until all islets are in the camera field of view at 10x magnification. Using cellSens software, capture brightfield images at approximately 12 ms exposure and darkfield images at approximately 400 ms exposure, each at 10x magnification. Ensure all islets are present in image. Save all image files.
SECTION: Islet Preparation and Loading
17. Ensure perifusion intake and air uptake pumps are off. Turn the stopcock on the air uptake lines so that the waste pathway is open, and close the stopcock on the outlet line at the fraction collector.
SECTION: Islet Preparation and Loading
18. Remove the chamber from its mounting, turn it upside down, and remove the red end piece (inlet). Remove and discard two thirds of the media from the chamber.
SECTION: Islet Preparation and Loading
19. Using a 1-mL micropipette, transfer the slurry of islets from the microcentrifuge tube to the perifusion chamber. Rinse the tube at least 3 times with baseline media and transfer to the chamber.
SECTION: Islet Preparation and Loading
20. Place the chamber back onto the mounting, and fill it up with baseline media until there is a convex meniscus. Tap the sides of the column lightly to dislodge air bubbles from the walls, and collect and discard any bubbles from the top of the meniscus.
SECTION: Islet Preparation and Loading
21. When all the bubbles have been removed, carefully replace the inlet plunger. 
 

SECTION: Islet Preparation and Loading
22. Turn the chamber right side up, and put it back on the mounting rack. Open the outlet line at the fraction collector and close the waste line on the air uptake line.
SECTION: Islet Preparation and Loading
23. Turn on the intake and air uptake pumps, set the fraction collector to 3 min and flip the collector arm so that it is over the first collection tube. Press “Start” on the collector to start the timer.
SECTION: Islet Preparation and Loading
24. Tighten column end pieces, and end fittings, and make sure there are no leaks. Lower the column mounting rack into the water bath, and tighten the clamp to prevent wobbling.
SECTION: Collection of Perifusate Fractions
26. Record each intake pump speed on the perifusion worksheet in the perifusion logbook.
SECTION: Collection of Perifusate Fractions
29. Restart intake pump until next secretagogue change.
SECTION: Collection of Perifusate Fractions
30. When perifusion is complete, cap the perifusion fractions and store at -20 °C.
SECTION: Collection of Perifusate Fractions
28. Move the media intake catheters from one secretagogue to the next, making sure to not tangle the tubing and that the catheter is resting on the bottom of the bottle.
SECTION: Islet Recovery and System Cleanup
37. Turn off intake and air uptake pumps, drain the water bath, turn off gas, and record the perifusion date, details, and any errors or problems in the perifusion logbook.
SECTION: Islet Recovery and System Cleanup
36. Rinse all lines, including waste lines, with deionized water at maximum pump speed for approximately 90 min.
SECTION: Islet Recovery and System Cleanup
35. Reassemble column and rinse all lines, including waste lines, with 10% bleach at maximum pump speed for approximately 15 min.
SECTION: Islet Recovery and System Cleanup
34. Rinse the chamber with deionized water and remove frits from chamber.
SECTION: Islet Recovery and System Cleanup
33. Carefully remove the blue outlet end piece from the islet chamber. Pipet the islets and media from the chamber into a 6-cm untreated tissue culture dish and rinse the chamber and the blue column end piece with 5 times with 1 mL each of baseline media into the dish. 
SECTION: Islet Recovery and System Cleanup
32. Raise the mounting rack from the water bath and wait approximately 2 min so that islets can settle to the bottom of the chamber.
SECTION: Islet Hormone Extraction
39. Using a pipette, transfer all islets from the center of the dish to a clear 1.5-mL microcentrifuge tube for islet hormone extraction.
SECTION: Islet Hormone Extraction
40. Centrifuge the tube at 200 rcf, 3 min and aspirate the supernatant using a pipette, being careful not to disturb the islet pellet. Place tube on ice.
SECTION: Islet Hormone Extraction
41. Prepare fresh acid alcohol for hormone extraction by adding 50 µL 12M HCl to 5.5 mL 95% ethanol.
SECTION: Islet Hormone Extraction
42. Centrifuge tube at 3000 rpm, 5 min and transfer three 50 µL aliquots of supernatant to 2-mL screwcap tubes. Store at -80 °C.
SECTION: Data Collection and Analysis
44. Use the custom Count and Measure algorithm to determine islet count and mean islet diameter. Split adjacent but discrete islets using the Manually Split Objects tool to get an accurate islet count and mean diameter measurements.
SECTION: Data Collection and Analysis
46. Perform insulin and/or glucagon assays on perifusion fractions and extracts.
SECTION: Data Collection and Analysis
45. Use the mean diameter measurements to assign islets to a diameter group using the chart provided (  ).
SECTION: Islet Preparation and Loading
15. Using a 200-µL micropipette, transfer all islets from the center of the dish to a clear 1.5-mL microcentrifuge tube for loading into perifusion chamber. 
SECTION: Preparation of Base Perifusion Media and Secretagogues
4.1. Use a vacuum-filtration system to filter media, transfer to a side-arm flask, and de-gas at 37 °C for at least 30 min.
SECTION: Preparation of Base Perifusion Media and Secretagogues
5.1. Wait 30 min and check glucose levels using a glucose meter.
SECTION: Preparation of Base Perifusion Media and Secretagogues
5.2. Reserve 30 mL 5.6 mM glucose media in a 50-mL conical tube for islet loading and unloading.
SECTION: Preparation of Base Perifusion Media and Secretagogues
6.1. Wait 30 min and check glucose levels using a glucose meter.
SECTION: Preparation of Base Perifusion Media and Secretagogues
8.1. 
Make 200 millimolar (mM) stock by adding the appropriate volume of DMSO (see Table 1).
 
 

 

SECTION: Preparation of Base Perifusion Media and Secretagogues
8.2. Dilute IBMX stock solution (prepared in step 8.1) to a final concentration of 100 micromolar (µM) by adding 100 µL IBMX stock solution to 100 mL 16.7 mM glucose media.
SECTION: Islet Hormone Extraction
41.1. Add 200 µL acid ethanol (prepared in step 41) to islet pellet and incubate at 4 °C 1440 min or for up to 1440 min.
SECTION: Islet Preparation and Loading
13. Prepare islet picking media (HBSS with 10% FBS): Remove and discard 50 mL from a 500-mL bottle of  , then add 50 mL  .