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I am interested in utlizing the FACS to identify cells undergoing apoptosis.
We have tried the Oncor Apotag Kit to label fragmented DNA and have had mixed.
Does anyone have experience with this system or any other kit/system to label
fragmented DNA for analysis by flow.?
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_/_/_/_/ _/_/_/_/ _/ _/ _/ _/Bruce J. Shenker Ph.D.
_/ _/ _/ _/_/ _/ _/_/ _/ shenker@biochem.dental.upenn.edu
_/_/_/_/ _/_/_/ _/ _/ _/ _/ _/ _/ (215)898 5959;FAX 573 2050
_/ _/ _/ _/_/ _/ _/_/ School Of Dental Medicine
_/ _/_/_/_/ _/ _/ _/ _/ THE UNIVERSITY OF PENNSYLVANIA
I presented a poster at the ISAC meeting in Lake Placid entitled "COMPARATIVE
STUDY OF FLOW CYTOMETRY AND IMAGE ANALYSIS IN SITU APOPTOSIS DETECTION OF CELLS
INDUCED BY HEAT SHOCK AND DEXAMETHASONE" using Oncor's ApopTag kits.(TdT
anti-digoxigenin fluorescein and peroxidase).
Initially I also had problems detecting DNA fragments following Oncor's
protocol,however, I overcame this dilemma by modifying a few steps describe in
Oncor's procedure. I have had pretty good results using human PBLs, mouse
thymocytes,and Jurkat cells.
I experienced massive cell loss after the 70%EtOH treatment during the wash
steps and I would therefore recommend starting with at least 2x106 cells/mL.
I've had good success limiting cell loss during the assay by using either a
microcentrifuge tube or 96-well V-bottom plate. This not only reduces cell
loss, but reagent use as well. Heat shocking Jurkat cells at 42oC for 1 hour,
and subsequently incubating at 37oC in complete media proved to be an excellent
control (eg. >30% positive over unstimulated control). Camptothecin and
dexamethasone are also very good inducers.
Good Luck!
O. Joseph Trask
Ohio State University
Comprehensive Cancer Center
Voice: 614/292-FLOW
Fax: 614/292-7335
e-mail: otrask.1@osu.edu
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