[cell biology work] apoptosis analysis

[Seung Jae Jeong]

TNFa/Fas mediated apoptosis

 

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.

 

 

 FACS analysis of apoptotic cells

 

1.     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.

2.     Centrifuge for 5min at 1500rpm and discard supernatant.

3.     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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