Annette, Enzymatic treatment may have an affect on surface antigens. We found that certain components of Collagenase D would selectively cleave surface antigens (we saw the greatest from the surface of leukocytes (unpublished data). We tried using trypsin inhibitors, but that did not help. Our final decision was consistency. For our preps, 30 minutes at 37 degrees with 400 U/mL collagenase D, then immediately dilute 10 fold with ice cold HBSS + 4% BSA + 1 mM EDTA (collagenase D is calcium dependent). We found very little cleavage right at 30 minutes, and if we were consistent with our timing, then preparations could be compared from day to day. That said, we were both mechanically (needle dissection) and chemically dissociating the tissue. We found either one alone lost a great number of the cells of interest (dendritic cells, in our case). Good luck. Kb -- Keith Bahjat, PhD Research Scientist Cerus Corporation Concord, California me@keithbahjat.com on 7/15/02 11:22 AM, Annette Byrne at AByrne@pcyc.com wrote: > Good morning Flow-ers ! > > > Just a quick query on single cell suspension prep for Flow: Am about to try > making cell suspensions from tumor and aortic tissue for Flow with fluorescent > membrane antibodies. My question is whether it's better to mechanically > disaggregate tissue or enzymatically (collagenase etc). Does enzymatic > tretatment have any negative effect on cell surface antigens ? > > Many thanks as always, > > Annette > Annette Byrne PhD > Scientist > Pharmacyclics > 995 E Arques Ave > Sunnyvale > CA 94085-4521 > USA > > Tel: 001-408-3283640 > Fax: 001-408-3283689 > e mail : AByrne@pcyc.com > www.pharmacyclics.com > >
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