| Earle’s balanced salt solution (EBSS) |
100% |
| HEPES |
10 mm |
| d-glucose
|
20 mm |
| Collagenase |
1.3 mg/mL |
| Trypsin |
0.60 mg/mL |
| DNase I |
0.05 mg/mL |
| Prepare EBSS in deionized water. Supplement EBSS with HEPES and d-glucose at the concentrations indicated. Adjust the pH to 7.4 and filter the solution by using a 0.2-µm-pore membrane filter.
Divide into 5-mL aliquots and store for up to 3 mo at 4°C. Add enzymes to the supplemented EBSS immediately before the start
of dissociation. (The concentrations of collagenase and trypsin must be empirically optimized with each new lot of enzymes.)
Filter dissociation medium with enzymes by using a 0.22-µm syringe filter (Millex-GV; Millipore #SLGV033RB).
|
