I have been getting very low yield from islet isolations from rats.
My procedure is as follows:
Inject the pancreas with 5mL Liberase (1 wunch unit/ml) in HBSS + HEPES through the CBD (decent inflation, atleast of the frontal part of the pancreas) and excise it to an Erlenmeyer flask with 5mL Liberase in HBSS + HEPES on ice (takes ~10 minutes).
Once all animals have been processed, I incubate for 21 minutes at 37C.
After incubation, I shake for 5 seconds and stop digestion using HBSS + 10% FBS. I sediment for 20 minutes and homegenize again using a wide-tipped pipette.
The tissue is pelleted and dissolved in 20mL Histopaque gradient. Spun at 380g for 20min at RT.
Wash twice in culture media and handpick islets.
Can anyone flaw my procedure or give me some clues as to what might cause very low yields in general? I've read somewhere that blood and fat tissues might inhibit digestion. Does anyone have any experience with this?
My current concern is that the islets are over digested.
Thanks in advance.