Apoptosis detection using PI on FACS - why can I not see a sub g1 peak? (Mar/13/2005 )

Hi all,

I have been trying for weeks to observe a sub G1 peak that corresponds to apoptotic cells after treatment with TRAIL. So far I have seen nothing but normal profiles (G1, S, G2). I have been fixing using the method below:

1.Fix in cold 70% ethanol.  Add dropwise to the cell pellet while vortexing.  This should ensure fixation of all cells and minimise clumping. 

2.Fix for at least 30 minutes at 4°C. Specimens can be left at this stage for several weeks. 

3.Wash x2 in Phosphate-citrate buffer.  Spin at 2000rpm and be careful to avoid cell loss when discarding supernatant especially after spinning out of ethanol. 

4.To ensure that only DNA is stained, treat cells with Ribonuclease.  Add 50 µl of 100 µg/ml RNase. 

5.Add 200 µl propidium iodide (50 µg/ml). 

6.Analyse by flow cytometry.

Can anyone explain why I am not seeing this peak in HCT116 cells treated with TRAIL (10ngml) over a time course of days ?

Thanks on advance
PJ

I used this technique some time ago, and the only difference I see with yours is that I fixed the cells in ethanol for 5 min @ -20ºC. Also, I always processed the cells inmediately, even thought the protocol says that you can stop at that step.
I guess you're recovering all the floating cells and that you're seeing apoptosis by other independent method. I don't know what to suggest, as this is quite a straightforward method. Maybe you could use a higher number of cells for your assays?
Sorry I could not be of more help. Good luck!

Start by collecting the media of the cells you wish to FACS. Your dead cells are mostly in the media. After trypsinizing cells add trypsinized cells to collected media. Spin at a speed higher than you would normally for HCT116 cells (apoptotic cells are light). Try around 3200 g, and it is extremly importnant NOT to use breaks on the centrifuge. Now, for all the folloiwng steps, centrifuge at 5000 rpm (on an eppendorf referigerated centrifuge). You should be able to see a sub G population.
Good Luck

Another detail that can be overlooked is the gate being used to measure your subpopulation of cells going into the cytometer. If someone was using it to strictly measure cell cycle before you, they may have gated out all the sub-G1 measurements.