Dear Flowers I am interested in finding apoptosis in CD4 and CD8 positive PBMCs using Apo-direct Tunel phoenix kit. Please kindly look at procedure and dot plots in attachment. I would be grateful if you could kindly send me your comments about the results. Is it correct or not? PBMCs were isolated from peripheral blood using ficol centrifugation. 1. I million Cells were stained with 10 micro liter CD4 or CD8 (PE) monoclonal antibody for 30 min at refrigerator. 2. Washed with PBS 3. Fixed in BD cell fix (10 times diluted in distilled water) for 15 min at room temperature. 4. Washed with PBS 5. Incubated in 0.1% triton X100, 4% inactivated FCS in PBS for 3-5 min at room temperature 6. Washed with wash buffer of the kit 7. Stained with DNA staining solution from the kit (1 hour at 37 C) 8. Washed with rinse buffer of the kit 9. Analyzed with FACScalibur Thanks a lot Kind regards Marjan Sincerely yours: Marjan Gharagozloo (Ph.D student) Department of Immunology Shiraz University of Medical Sciences Shiraz, Iran PO Box:71345-3119 Phone:+98-913-1100447 Fax:+98-311-6259769 This attachment - 'tunel CD8.jpg' - 20.68 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/e6261163e789c3ca33924b4990316f074674d74a.jpg This attachment - 'FSCSSC.jpg' - 29.80 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/168e44db0c1705b9585620916b9c1d6c8263bf4c.jpg This attachment - 'tunel CD4.jpg' - 19.95 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/d5f285c961e0c37599a0eb47060cfc89723fef32.jpgReceived on Wed Dec 22 12:38:00 2004
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