[Obi-protocol-application-branch] Apoptosis by detection of dna fragmentation

[Alan R. <ala...@gm...>]

From: http://www.icms.qmul.ac.uk/flowcytometry/uses/apoptosis/ 
dnafragmentation/index.html
DNA Fragmentation

There are certain characteristics of apoptotic cells that can be  
identified and used to detect apoptotic cells in an otherwise healthy  
population of cells. Technically the easiest characteristic to detect  
is loss of DNA from permeabilised cells due to DNA fragmentation.  
When cells are permeabilised, for example by 70% ethanol, the  
fragmented 182bp DNA multimers leak out of the cell. The result is a  
population of cells with a reduced DNA content. If the cells are then  
stained with a DNA intecalating dye like propidium iodide, then a DNA  
profile representing cells in G1, S-phase and G2M will be observed  
with apoptotic cells being represented by a sub G0/G1 population seen  
to the left of the G0/G1 peak.

SubG1 Protocol

1.  Harvest cells washing in PBS.
2.  Fix pelleted cells in ice-cold 70% ethanol by adding with a  
pasteur pipette on a vortex. Leave cells at 4oC from 30 mins to a week.
3.  Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in  
Phosphate Citrate PBS buffer (0.1M Citric Acid pH 7.8).
4.  Add 50 ml RNAse (100 mg/ml Sigma) and incubate at RT or 37oC for  
15 mins.
5.  Add 200 ml of PI (50 mg/ml Sigma P4170).
6.  Analyse by flow cytometry collecting 25,000 events per sample.


More protocols at http://www.icms.qmul.ac.uk/flowcytometry/uses/ 
apoptosis/ e.g.

Annexin-V - Cell membrane changes
In normal cells, phosphatidylserine (PS) residues are found in the  
inner membrane of the cytoplasmic membrane.  During apoptosis, the PS  
residues are translocated in the membrane and are externalised.  In  
general, though not always, this is an early event in apoptosis and  
is thought to be a signal to neighbouring cells that a cell is ready  
to be phagocytosed.  Annexin-V is a specific PS-binding protein that  
can be used to detect apoptotic cells.  Annexin-V is available  
conjugated to a number of different fluorochromes.

Annexin V Staining Protocol

1.   Pellet cells, including any cells in supernatants from adherent  
cell cultures.
2.    Resuspended in 400 ml of Becton Dickinson (BD) Annexin V  
Binding Buffer (Cat. No.556454).
3.   Add 2 ml Annexin V-FITC (BD, Cat.No. 556420) or 2 ml Annexin V- 
AlexaFluor-647(Invitrogen Cat. No. A23204.
4.   Incubate at RT for 15 mins
5.   Add viability dye PI (5 mg/ml) or DAPI (200 ng/ml)
6.   Collect 10-20,000 events.

-Alan