Calcium Free Islet Dispersion Fluid

1) 138 NaCl ( 1.58 g / 200 mL solution)

2) 5.6KCl ( 0.08 g / 200 mL solution)

3) 1.2 MgCl₂ ( 0.016 g / 200 mL solution)

4) 5 HEPES ( 0.248 g / 200 mL solution)

5) 3 glucose ( 0.108 g / 200 mL solution)

6) 1 EGTA ( 0.076 g / 200 mL solution)

7) 1 mg/mL albumin

adjust pH to 7.3 with NaOH

1. pick islets into 35 mm dish with 2 ml ‘dispersion fluid’

2. repeat step 1 (pick into a new dish with 2 ml ‘dispersion fluid’)

3. pick all islets in a very small volume (~100 ul) into a 15 ml falcon tube

4. incubate at 37 C for 11 minutes

5. triturate gently until solution becomes cloudy and visible islets disappear

6. add ~0.5- 1 ml of regular islet culture media (DMEM+FBS+pen-strep- the volume used will depend on the number of islets being dispersed)

7. mix and plate as drops in the centre of 35 mm dishes (dishes may be pre-coated with high molecular weight poly-lysine to aid in cell sticking)

8. after at least 2 hours, add 1-2 ml of DMEM culture media