Hi Joy, We have used a method using enzymes dissolved in RPMI\10%fcs for 7 or 8 years, with no apparent problems. The method uses 10mg deoxribonuclease + 10mg collagenase type 1A dissolved in 10 mL RPMI/10%FCS/hepes/PSA. To approx 1 mL of this enzme solution the tissue is added and gently minced using a two scalpel technique. The resulting cell suspension is placed in a 17x100 plastic tube along with the remaining small tissue fragments. The cells can be rocked in a 37 degrees incubator for up to 3 hrs with no apparent affect on cell viability. Cells are washed in PBS x2, and resuspended in fresh RPMI\10%FCS. Viability after 30 minutes is usually >95%. Cases showing fibrosis, and where the viability of the cells may be a problem, we have tried just deoxyribonucease, mince, leave for 15 minutes at room temperature on a mixer and then procede with rest of the method. Granted the viability will still be a problem, and you will have to assess your results on case by case basis along with the histology of the tissue. I hope this may be of some use. Paul Paul Harris Flow Cytometry Lab Dept. of pathology St. Joseph's Health Centre 268 Grosvenor St. London, On. Ca. Fax (519) 646 6088 ph (519) 646 6100 ext 5918 Email paulh@stj.stjosephs.london.on.ca.
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