Protocol
Collagenase Preparation Weigh out the collagenase into a 50 ml conical and add isolation buffer to make the final concentration, as indicated in the table below. Dissolve the collagenase by vortexing.(12 mice + 2 extra mice = 14 mice x 5 ml/mouse = 70 ml total collagenase solution)(70 ml x 0.5 mg/ml or 0.3 mg/ml = 35 mg or 21 mg of collagenase P total) <table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP: #ddd 1px solid; BORDER-LEFT: #ddd …
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Dithizone | reagent | Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 “m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed. | ||
| PBS | Buffer | |||
| HBSS | Buffer | |||
| Isolation soln. | Buffer | HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4 | ||
| Wash soln. | Buffer | Isolation buffer containing 1mM CaCl2 | ||
| Collagenase P | Boehringer Mannheim-Roche | 1213873 | ||
| Dissection tools | Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue) | |||
| Syringe | sterile, 5ml, 1/mouse. | |||
| 30 G needles | sterile, 1/syringe | |||
| siliconized glass vials w/Teflon cap | Fisher | 213-018-54 | sterile, 1/mouse | |
| large sink strainer | sterile | |||
| 1000 ml Beaker | sterile | |||
| Gauze pads | 4×4, 2/mouse | |||
| plastic eye dropper | sterile | |||
| Mice | Animal |
Citazione di questo articolo
Szot, G. L., Koudria, P., Bluestone, J. A. Murine Pancreatic Islet Isolation. J. Vis. Exp. (7), e255, doi:10.3791/255 (2007).