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| Fluorescence Activated Cell Sorting (FACS analysis) |
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| This protocol is used for preparation of cells for FACS analysis to understand the
cell cycle state of the cells. |
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1. Spin cells at 2000 rpm for 5 min and remove the supernatant.
2. Re-suspend cells in 1ml PBS chilled.
3. Spin cells at 2000 rpm for 5 min and remove the supernatant.
4. Re-suspend pellet in 1mL 2N HCl + 0.5% Triton X-100 chilled. Incubate at room
temperature for 30 min.
3.44 ml concentrated HCL (11.6M)
16.56 ml distilled water
1 ml 10% Triton X-100
5. Spin cells at 2000 rpm for 5 min and remove the supernatant.
6. Re-suspend cells in 1ml 0.1M Sodium Borate (Borax) chilled. This step is to neutralize
the acid.
7. Spin cells at 2000 rpm for 5 min and remove the supernatant.
8. Re-suspend pellet in 1ml Tween 20/1% BSA/PBS solution chilled.
9. Spin cells at 2000 rpm for 5 min and remove supernatant as best one can.
10. Add 30 ul anti-BrdU (FITC) directly on top of pellet. Incubate at room temperature
for 30 min.
11. Add 1ml Tween 20/1% BSA/PBS solution chilled and re-suspend the pellet.
12. Spin cells at 2000 rpm for 5 min and remove supernatant.
13. Re-suspend pellet in 1ml Tween 20/1% BSA/PBS solution chilled.
14. Spin cells at 2000 rpm for 5 min and remove supernatant.
15. Re-suspend cells in 0.5ml PBS/Propidium Iodide/RNase chilled.
100 ul 1mg/ml propidium iodide
20 ul 10 mg/ml RNase
20 ml PBS
16. Conduct FACS analysis
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| Date uploaded: 03-05-2011 |
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