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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web