Meenakshi, Actually, we use anti-FAS to instruct cells to go into this destructive process. 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 1640 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 µg/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°C for 2 h to 3 days. TdT Assay (Pat Blair): Following a single wash in HBSS, samples were resuspended in 50 µl of a terminal deoxynucleotidyl transferase (TdT) reaction mixture (0.1 M cacodylic acid, 1 mM CoCl2, 0.1 mM dithiothreitol and 50 µl BSA) containing 0.5 nM biotin 16-dUTP (Boehringer, Indianapolis IN) and 10 units of TdT (Boehringer) for 30 min at 37 °C. After a wash in HBSS, 2.5 µg/ml FITC-avidin (Gibco Life Technologies, 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 (Coulter Inc., Kindal FL) flow cytometer. Five to ten thousand cells were enumerated using the parameters of log ninety degree scatter versus log-FITC intensity. Apoptotic cells were defined as FITC-positive cells with a low forward angle 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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