Reagents

All reagents must be sterile.

Hanks balanced salt solution without calcium and magnesium (HBSS)

Collagenase (crude, Type I or IV) (Note 1: Dissolve the contents of the entire bottle in HBSS to give a concentration 10x the working concentration required. Filter-sterilize it through 0.45^ and 0.22^ filters using the plastic syringe. Dispense into aliquots suitable for the amount of tissue to be disaggregated and store at -20 °C. Note 2: Do not use serum in the diluting medium because it is difficult to filter-sterilize when the protein content is high. Note 3: Follow the same procedure to prepare a stock solution of dispase.)

Incubation medium (see Protocol 3.1 for notes on storage medium) containing 10% serum (e.g. fetal calf or calf)

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