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Actually, we use anti-FAS to instruct cells to go into this destructive p=
rocess. =
See procedure below.
Stephen P. Perfetto
Department of HIV Disease Prevention
Walter Reed Army Institute of Research
1600 East Gude Drive
Rockville, MD. 20850
Positive Control for Apoptosis:
1. J3 Jurkat cells were cultured for 24 h at 1 x 106 cells/ml in RPMI 164=
0 =
supplemented with 10% FCS (T cell medium) in 24-well plates. =
2. These cells were washed twice in HBSS and treated for 24 h with 0.5 =B5=
g/ml =
human anti-Fas antibody (Upstate Biotechnology Inc. Lake Placid NY).
3. After this incubation samples were washed in HBSS and cell pellets =
resuspended in 70% ethanol and stored at -20=B0C for 2 h to 3 days. =
TdT Assay (Pat Blair):
Following a single wash in HBSS, samples were resuspended in 50 =B5l of a=
terminal =
deoxynucleotidyl transferase (TdT) reaction mixture (0.1 M cacodylic acid=
, 1 mM =
CoCl2, 0.1 mM dithiothreitol and 50 =B5l BSA) containing 0.5 nM biotin 16=
-dUTP =
(Boehringer, Indianapolis IN) and 10 units of TdT (Boehringer) for 30 min=
at 37 =
=B0C. After a wash in HBSS, 2.5 =B5g/ml FITC-avidin (Gibco Life Technolo=
gies, =
Gaithersburg, MD) was added to a staining solution (4 x SSC, 0.1% Triton =
X-100 =
and 5% non-fat dry milk) and samples were incubated for 15 min at room =
temperature. =
Flow Cytometric Assay:
Samples were analyzed by cytofluorometric analysis using an ELITE-ESP (Co=
ulter =
Inc., Kindal FL) flow cytometer. Five to ten thousand cells were enumer=
ated =
using the parameters of log ninety degree scatter versus log-FITC intensi=
ty. =
Apoptotic cells were defined as FITC-positive cells with a low forward a=
ngle =
and low ninety degree scatter. Viability based on trypan-blue exclusion =
confirmed the reliability of the TdT-based assay.
_________________________________________________________________________=
______
Subject: OKT3
From: Meenakshi Roy <meena@macsun.stanford.edu> at Internet_Gateway
Date: 1/4/97 5:32 PM
Hi,
Does anyone know where I can buy purified OKT3? There are many other
anti-human CD3 antibodies on the market, but I am going to use this
antibody to induce apoptosis. I dont know whether any of the other
antibodies have been used to induce apoptosis
I would appreciate any info, advice etc,
Dr. Meenakshi Roy
Stanford University.
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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;
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