Problems about detection of apoptotic cells using Annexin V/PI staining - (Feb/18/2011 )

I am new to flow cytometry analysis and no one in my department has ever done flow cytometry analysis on living cells before. Sorry if my questions seem stupid^^

I am working on adherent cells. But they generally detach from the culture dish after apoptotic induction with our chemical inducing agents so I just wash them out without trypsinization. What I am doing now is: collect the cells > centrifuge to remove medium and add PBS for washing > centrifuge and discard PBS > add annexin V binding buffer, Annexin V-Alexa and PI > incubation at room temperature for 15 min > examination of samples under fluorescence microscope (not started FC yet).

(1) Does centrifugation (say 150g, 4min for 2-3 times) and pipet up-and-down cause more damage to the cell membrane, resulting more-than-expected number of PI +ve cells?
I know that untreated, intact control cells can tolerate centrifugation and pipet up-and-down quite well and they won't become PI +ve after those processes. But I am doubtful about the induced apoptotic cells as they seem so vulnerable. I see quite a few protocols of commercial Annexin V/PI kits require centrifugation (and of course resuspension) of cells so it should be OK?

(2) Can I fix the cells after Annexin V/PI staining with 4% paraformaldehyde? Does it cause loss of Annexin V signal? I am now using kit from Invitrogen.

(3) Should I use PBS at room temperature or cold PBS to wash the cells before addition of Annexin V binding buffer? I tried cold PBS according to protocol from Invitrogen to wash the apoptotic cells that are still attached to the culture dish and quite a lot of cells "lysed" immediately (1-2 large grey circles attached to the edge of the cells, similar to leakage of cell content after necrosis)

(4) What is the reason for keeping the cells on ice after staining before FC? To keep the fluorescent signals or to keep the cells alive? Does it help keep the cells remaining PI -ve or would the cold temperature cause more cells to become PI +ve? When I examine the stained cells with microscope, the Annexin V +ve/PI-ve cells turned PI+ve after a few minutes. Is that normal?

(5) Can I add Hochest into the staining solution so that I can use Hochest as discriminator for event collection during FC? Or should I just set forward scatter as discriminator for event collection?

some thoughts

1-any manipulation will damage but since the control cells also go through the same procedure, its fine. One can be gentle or try to optimise it so that you are gentle. Your protocol sounds fine.

2- I know some people do fixs cells after staining. I donot know if there is loss but I would expect some loss of signal. we use kit from BD, it works quite nicely.

3- we use PBS without ca and mg) at room temperature.

4- its good that the signal turns from Pi-negative to positive. So the cells actually are going through the whole cell death process

good luck

1. Yes, it could but not significantly.
2. Definitely, NO!
3. Use RT
4. The reason is to minimize the non-specific binding and AnnexinV detachment.
5. Yes, you can. But check the compensation.