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| Cell cycle analysis:
| | [[HD:cell cycle.pdf]] |
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| Plate cells according to time points. Confluency=30%
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| After 24 hrs of culture in complete media, serum starve the cells. (Medium-DMEM+7.5% BSA)
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| To synchronize G0/G1 phase, let the cells grow for 48-72 hrs.
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| After the set time, harvest the cells at different time points (intervals of 6h, 12h, 18….) and fix in
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| chilled 70% ethanol.
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| Fixation:
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| Trypsinize the cells and wash with PBS. Transfer cells to flow tubes in 100uL PBS.
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| While vortexing, add chilled (-20 C) 70% ethanol dropwise (~2 mL) to avoid clumping. This can be
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| stored at -20 C for 2-3 weeks.
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| Annexin V and PI staining:
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| After harvesting, spin down by washing with PBS containing 2% FBS and 1% HEPES for 10 mins at
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| 2000 rpm.
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| Discard the supernatant and dissolve the pellet in 400 uL PBS (2% FBS and 1% HEPES). Pass this
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| through 40um cell strainer.
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| To this add 15 ul annexin V and incubate for 1 hr at 37 C. Wash with cold PBS buffer once. Resuspend
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| in 200 ul PBS buffer.
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| Add PI (25 ug/ml) to the same tube and keep in dark at 37 C for 30 mins.
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| Store at 4 C covered in foil until flow acquisition.
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