Greetings, I am having some trouble with my attempts at labeling cells (HL60) with BrdU. I have not been able to see any labeling with BrdU-FITC antibody. I am using the following basic protocol: 1. Incubate cells for 30 min/37*C with 20µL/mL of 10mM BrdU Stock. (Flasks are foil wrapped to avoid light exposure.) 2. Spin cells, remove media supernatant. 3. Wash 2x with cold PBS. 4. Resuspend pellet and fix with cold 70% ETOH and store at 4*C in the dark. (Usually left overnight.) 5. Centrifuge cells, remove ETOH supernatant. 6. Resuspend pellet and add 3mL 4N HCl and incubate 30 min/RT. 7. Centrifuge cells, remove HCl supernatant. 8. Resuspend pellet and wash with PBS. 9. Resuspend pellet and wash with 0.5%Tween 20 solution+PBS . 10. Centrifuge cells, remove Tween 20 supernatant. 11. Add 10µL anti-BrdU-FITC and incubate for 30min/RT/dark. 12. Wash 2x with cold PBS. 13. Resuspend pellet and add 1mL of PI in PBS. I have seen other protocols and they are more/less the same. Can anyone share some insight on my problem. Is there something that I am missing? Thanks. Richard **************************** Richard A. Salinas Flow Cytometry Laboratory Dept. of Radiation Oncology UTHSCSA Texas Research Park (210) 677-3882 rsalinas@saci.org
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