Many people use Acridine orange. It apparently can detect DNA damage associated with apoptosis. All you do is make an acridine orange solution (I forget the concentration) then Let the tissue/cells soak in it for 15 to 20 minutes, remove it, then analyze the results under a flurorecscent microscope. The cells that are undergoing apoptosis will fluoresce while the nonapoptotic cells will not.

Does anyone know of a rapid, simple method of detecting Apoptosis in adherent cell lines?

could you please explain how the assay works because I don't understand. does it discriminate between apoptotic and necrotic cells?

how rapid?

and

why it has to be rapid ditection?? (are there any specific reasons?)

also, tell us what kind of things are available in your lab (i.e. FACS)

Hi all,

i generally use DAPI staining to study apoptosis on adherent cells:

-cells are deposited on coverslipes in a 6d plate and treated with apoptotic agents
-wash the cells twice with PBS
-fix the cells with MeOH 5min at RT and let dry for a moment
-stain with 500 µl DAPI at 0.5µg/ml for 30min at RT in the dark
-wash twice with PBS
-mount on slide and visualize cells under microscope with a 360-370nm excitation light and 420-460nm emission filter; apoptotic cells appear with characteristic condensed and fragmented nuclei

Seb_

One quick method that will give you a general idea of apoptosis in your cells is to stain your cells with propidium iodide and look for the sub-G1 population using FACS. I can give you a protocol if you would like

you can also use the DiOC6(3) dye (see attached pdf)
after a wash, incubate the cells in a 40nM solution for 15-20min at 37C
then directly analyze apoptosis by flow cytometry

Seb_

Hi,
I have actually been looking into studying apoptosis myself...and find that using acridine orange/eithidium bromide and performing flourescence microscopy is a very good option. However, my cells are not asherant but in suspension. Also, I want to make a cell count ratherthan a qualitative analysis, so is there a special kind of slide available that can be used for counting cells in this situation? I heard of 6d-plates but that seems like an expensive option and probably is for qualitative analysis, not quantitative.
Penny