Cell culture facility (Biosafety level 2 is required when working with human material, while islets of rodent origin can be manipulated in Biosafety level 1)
Laminar flow hood
24 or 48-well cell culture plates (e.g., Cell Star, Greiner Bio-One #662160 and #677180)
Round 8 or 12 mm diameter and 0.17 mm thickness borosilicate glass coverslips (Electron Microscopy Sciences #72230-01)
Or Coverglass-bottom Fluorodishes 35mm (World Precision Instruments #FD35-100)
Or Coverglass-bottom MatTek dishes 35mm (MatTek Corporation #P35G-0.170-14-C)
Glass beaker (100-150 ml)
Research pipettes with 1000 μl, and 200 μl pipette tips with filter
Standard 15-ml polypropylene lab tubes (e.g., Falcon 15-ml tubes with conical bottom and blue screw cap; Falcon, #352070)
Table top centrifuge for use with 15-ml tubes
Water bath at 37 °C
Incubator at 37 °C and 5% CO2
Disposable bottle filter (0.22 μm) (e.g., Vacuum Filtration 500 “rapid”-Filtemax, TPP, #99500)
Hemocytometer for cell counting
Optical microscope with 10x and 20x objectives
• Pancreatic human or rat islets: Rat islets isolated from P5 rats (we recommend using the protocol described in Kanaani, et al., (2015) and pancreatic human islets obtained from pancreatic donors. Important: Viability and purity of islets are key factors in obtaining monolayer cultures of high quality, viability and functionality.
• Laminin and collagen IV solution: Thaw (on ice) a laminin or collagen IV aliquot (stock solution 1 mg/ml) for preparation of rat or human islet monolayer, respectively. Caution: Laminin/collagen IV can gel if left at room temperature.
• Culture medium: Prepare neuronal culture medium by supplementing MEM medium with 5% FBS, 1x B-27, 1% P/S, HEPES 10 mM, Glutamax 1x, Na-pyruvate 1mM, and glucose to a final glucose concentration of 11 mM.
For 50 ml of medium:
MEM 44.3 ml
FBS 2.5 ml
Pen/Strep 0.5 ml
B-27 1.0 ml
Pen/Strep 0.5 ml
HEPES 1M 0.5 ml
Glutamax 100x 0.5 ml
Na-pyruvate 100mM 0.5 ml
Glucose 25% 0.2 ml
• Glass coverslips Transfer the number of glass coverslips needed into a glass beaker and clean by sonication in 100% ethanol. Remove ethanol, cover the beaker with aluminum foil and sterilize in autoclave.