| 1. |
| Pulse actively growing cells in a tissue culture flask for one hour with 10 � 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 �. Incubate 20 minutes at RT. |
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| 4. |
| Aliquot 100 � 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 � contains the optimal concentration. Resuspend cell pellet in 50 � 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 � contains the optimal concentration. Resuspend cell pellet in 50 � 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 �/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. |
