RE: apoptosis

Tom Mc Closkey (
Wed, 23 Apr 97 11:38:32 PDT

--- On Tue, 22 Apr 1997 11:30:35 SAST-2 "Hughes, EJ, Jane, Ms"

>We are setting up an apoptosis assay using FITC anti BrdU

>1. Should one use an Isotypic control.

I think the best control for this assay is to prepare the cells you
are testing in an identical manner, just leave out the enzyme.

>2.I want to immunophenotype first, should I lyse the RBC after
>immunophenotyping and before DNA cleavage. (I wasn't sure from the
>method whether Triton would lyse the cells as RBC lysis is not given
>as a step)

Yo u may need to try lysing at different points in the assay to see
which works best. Another option is to use Ortho's reagent Permaefix which
works witht eh TUNEL assay and has been reported to lyse rbc.

>3. What are the options for positive controls.

The easiest is to use a cell line which is irradiated or treated
with a known apop inducing agent. these can be prepared in batch and fixed
and kept for some time. Another option is that Phoenix Flow Systems has
negative and positive control cells available.

Good luck,

Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Biomedical Research Center
350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office]; 516-562-1135/4641 [lab]
4/23/97 11:45:04 AM

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