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.