Apoptosis detection of adherent cells - Annexin V? (Aug/06/2009 )

Do you know a good method to detect apoptosis on adherent cells? I've read on the datasheet of BD Annexin V Apoptosis detection kit "Investigators should note that FITC Annexin V flow cytometric analysis on adherent cell types (e.g HeLa, NIH 3T3, etc.) is not routinely tested as specific membrane
damage may occur during cell detachment or harvesting." Do you know an alternative method?

intracellular staining of cleaved caspase 3.

Tunel assay.

miBunny on Aug 9 2009, 08:20 PM said:

wow, thanks. but do you know if it is cheaper/more expensive than Annevin V?
I have received these suggestions for using Annexin V on adherent cells: "we suggest using trypsin without EDTA to detach cells, as Annexin V staining requires calcium. In literature, you might find that cells were detached and harvested using versene or PBS + EDTA. In this case, it is very important to wash well the cells, to discard all the EDTA, before performing the staining. It is also advisable to check the integrity of the cells with Trypan Blue before performing the assay".
what do you think about?

also, since I already have PI solution, is it possible to buy separately Annexin V-FITC solution and the so-called binding buffer? Do you eventually have a protocol?

Thanks

Annexin is by far the cheapest option! Intracellular caspase 3 is the most accurate option and TUNEL is the trickiest option.

For annexin, the trick will be to trypsinze or EDTA them off gently and wash well. The problem is that most people beat their cells up when they trypsinize them and there is membrane damage.

You can definitely buy Annexin-FITC by itself (BD, Ebiosciences, and Sigma all carry it and probably lots of other complanies). I haven't used the binding solution in years! I do these stainings in my cell culture media and run them on the FACs. There is more than enough calcium for effective binding.

My protocol is:
Cells are in 100 uL media at room temperature. Add Annexin-FITC and 7-AAD (or PI) directly to cells and incubate 5 minutes. Analyze on FACs machine (no washing). I usually get these solutions from BD and use them at 5 uL each per sample.

miBunny on Aug 11 2009, 04:02 AM said:

I use this exact same protocol except with a 15min incubation step.

Just a thought - at what point do cells lose adherence due to their ongoing apoptosis? I mean, if you are only looking at apoptosis in adherent cells you may be getting a very low value against true apoptosis as all the apoptotic cells will be washed off? Are you sure this is what the experiment needs?

miBunny on Aug 11 2009, 04:02 AM said:

thanks, I was very confused because I received very different suggestions. And also, there are papers with questionable data ( like Van Engeland et al. , Cytometry 1996) where they show a 56% of cells AnnexinV+ cells in an exponential cell culture after detaching with conventional trypsinization.
so, if I understood well, the cheapest option for me would be to :
1 Detach cells with trypsin alone, gently and without beating the plate ( that in the case of my cells would be leave 3 minutes with trypsin at 37 degrees, I think)
2 harvest cells and wash 2 times
3 resuspend cells ( how many?) in culture media ( like RPMI 10%FCS?) and add Annexin V-FITC and PI ( can you please tell me wich concentration?)
4 incubate 5 minutes at RT
5 analyze without washing

is everything correct?

thanks

in this paper "Van Engeland et al. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture, Cytometry 1996" they suggest to stain adherent cells with Annexin V-biotin, detach with scraping and then add streptavidin-FITC. what do you think?

If I was you, I'd collect all the suggestions and ideas and try them all. That's the best way to work out a new technique, a good old fashioned head to head comparison!

miBunny on Aug 11 2009, 04:02 AM said:

I´m also curious about the media since you get a very bad signal when running FACS in cell culture media like RPMI. Do you mean FACS buffer (i.e PBS/%FBS or FCS)?
Best regards carmen

I agree with the statement posted above...it is possible that by detaching cells from the plates, by using Tripsin or cell scraper, it is possible to damage the cells.

Personnally, I know another alternative method: cels labelling of cells with Annexin-V-FITC directly in culture (after the treatment of the cells with the drugs or other agents) and then observing the cells under a fluorescence microscope (after washing with PBS the monolayer to wash away the unbound dye) to observe annexin-V attached to the surface of the cells.

Obviously, by using this method, you cannot quantify the amount of Annexin-V that binds the surface of the apoptotic cells...