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FACS analysis of apoptotic cells
1. transfect empty vector and p43 DNA(1ug;12well, 2ug;6 well) into target cells and incubate for 20 hrs at 5% Co2, 37 degree.
2. change with serum free media and add apoptotic mix(10ng of TNFa + 10ug/ml of cycloheximide or 1/1000 of Fax + 10ug of cycloheximide in serum free media).
3. culture for 1-2 hrs and watch morphological change & make a photo.
from step 3, remove media and trypsinize to detach the cells, and wash with 1XPBS.
Centrifuge 1500rpm for 5min and resuspend with 1XPBS. Add 10% formaldehyde to the
final conc 5% and fix for 1 hr.4 Centrifuge for 5min at 1500rpm and discard supernatant.
5. resuspend the cells with 1X PBS. Stain with 50ug/ml of Propium Iodide(stock ; 5mg/ml) and wrap with foil to protect from light and analyze with FACS.
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