| | 1. | | Pulse actively growing cells in a tissue culture flask for one hour with 10 µM BrdU (Sigma, Cat. No. B5002). |
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| | 2. | | Pour contents of tissue culture flask into a centrifuge tube. Centrifuge 10 minutes at 400 x g (all centrifugation steps) are performed at 400 x g, at RT). Aspirate supernatant. Loosen pellet by tapping tube. |
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| | 3. | | While vortexing, add ice cold 70% ethanol to cells, dropwise, to a final concentration of 1 x 10 6 cells/100 µl. Incubate 20 minutes at RT. |
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| | 4. | | Aliquot 100 µl into each test tube (12 mm x 75 mm). Wash with 1 ml wash buffer. Centrifuge 5 minutes. Aspirate supernatant. Loosen pellet. |
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| | 5. | | Resuspend pellet in denaturing solution. Mix well. Incubate 20 minutes at RT. NOTE: Denaturing solution must be made fresh. |
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| | 6. | | Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant. |
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| | 7. | | Resuspend pellet in 0.5 ml 0.1 M sodium borate (Na 2 B 4 O 7 ), pH 8.5, to neutralize any residual acid. Incubate 2 minutes at RT. |
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| | 8. | | Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant. |
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| | 9. | | Add primary antibody: dilute anti-BrdU monoclonal anitbody (Pharmingen Cat. No. 33281A) in dilution buffer, such that 50 µl contains the optimal concentration. Resuspend cell pellet in 50 µl of the diluted antibody. Incubate 20 minutes at RT. |
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| | 10. | | Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant. |
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| | 11. | | Add secondary antibody: dilute FITC-conjugated goat anti-mouse Ig (PharMingen Cat. No. 12064D) in dilution buffer, such that 50 µl contains the optimal concentration. Resuspend cell pellet in 50 µl of the diluted antibody. Incubate 20 minutes at RT. NOTE: Eliminate this step when using FITC-conjugated anti-BrdU (Cat. No. 33284X) |
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| | 12. | | Add 1 ml wash buffer. Mix well. Centrifuge 5 minutes. Aspirate supernatant. |
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| | 13. | | Resuspend pellet in 0.5 ml propidium iodide (10 µg/ml in PBS). Incubate 30 minutes at RT, protected from light. |
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| | 14. | | Analyze the cells by flow cytometry, exciting at 488 nm and measuring the BrdU-linked green fluorescence (FITC) through a 514 nm bandpass filter and the DNS linked red fluorescence (PI) through a 600 nm wave-length filter. |
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| | 15. | | Following analysis, flush flow cytometer for 10 minutes with 10% bleach and 5 minutes with dH 2 O. |
