Pancreatic content measurement
Reagents:
- Acid ethanol (0.18 M HCl in 70% ethanol)
- 375 ml absolute ethanol
- 117.5 ml ddH2O
- 7.5 ml concentrated HCl
- store at 4C
Pancreatic insulin content measurement is a quick but relatively inaccurate approach to assay beta cell mass. Under different metabolic conditions, animals may decrease expression of insulin while beta cells are intact. The low pancreatic insulin content may be misleading. However, the approach may be informative especially combined with immunohistochemical staining.
1. Isolate pancreas or pancreatic remnant, trim off fat and connective tissues.
2. Get the wet weight (in mg) of each pancreas.
3. Place pancreas in 3 ml ice cold acid ethanol in 16 X 100 mm (or 17 X 100 mm) round-bottom tube, keep on ice.
4. Homogenize pancreas with Polytron or homogenizer.
5. Rinse the homogenizer with 3 ml acid ethanol, and add to the tube of tissue homogenate (6 ml in total by this step).
6. Store overnight at 4C.
7. Centrifuge at 2400 RPM for 30 min at 4C.
8. Transfer the supernatant to a 15 ml conical tube, store at -20C.
9. Re-homogenize the pellet with another 3 ml ice cold acid ethanol as step 4-5.
10. Leave the homogenate at 4C for overnight.
11. Repeat Step 6 & 7 (centrifugation, transfer supernatant into conical tube from step 8. You should have ~12 ml extract by here).
12. Re-homogenize pellet with 3 ml ice cold acid ethanol and leave at 4C overnight (not necessary to rinse the homogenizer this time).
13. Repeat centrifugation and put supernatant into the same conical tube (now you should have ~15 ml of extract).
14. Freeze at -20C until assay.
15. Before assay, warm extract to 37C, mix by vortex, and dilute by 1:500-1000 in ELISA assay buffer and take 10 ul for measurements.