this may help.

Before commencing any study of apoptosis using flow cytometry, you should, whenever possible, first establish the presence of these cells visually and should not rely on the various flow cytometric assays. Apoptotic cells were originally identified by the characteristic changes in their nuclei; this is still the best method of confirming their identity. Nuclear changes can be observed by staining with a DNA dye and using fluorescence microscopy.

The following features of the apoptotic cascade can be observed using flow cytometry.

Expression of proteins involved in apoptosis

Activation of caspases

Changes in the mitochondrial membrane potential

Changes in the plasma membrane

Cell shrinkage

Chromatin changes

DNA degradation. thanks

Hi Touat Zahia,

in my experience you can use Annexin V apoptosis detection kit first, and/or Tunel assay detection kit. 

Chromatin changes is visible in fluorescence microscopy also using a marker such as Hoecst or TO-PRO 3.

Good luck

Lorena

I recommend using at least 2 methods to assess apoptosis. All of the assays available can give confusing results, if you have a confirmatory assay then you are likely not to be misled.

Beware of the annexin V propidium iodide assay. This relies on annexin V binding to externalized phosphatidyl serine on the plasma membrane (Annx V positive) and on cells excluding PI (PI negative). PI shows that the membrane is intact-- as it should be in early apoptosis. If the cell is necrotic or the membrane is damaged, the cell will be Annx V positive-- stain has access to intracellular Phosphatidyl serine; same cell will also be PI positive-- stain has access to intracellular nucleic acids. AnnxV-positive and PI-negative cells are early apoptotic cells. AnnxV-positive, PI-positive cells may be necrotic, may be late apoptotic-- hard to know.

I have not had a good experience with AnnxV by microscopy. Flow cytometry is better. Look at nuclear morphology using a DNA stain like DAPI. Condensed, fragmented nuclei are consistent with apoptosis. Include a positive control, possibly staurosporine. Low levels of apoptosis (5-10%) are expected in culture. Express death as the percent apoptotic nuclei out of total nuclei. Consider looking at this 2009 paper: PMID:19373242.

Fluorescent caspase activity measurements are available and are generally reliable. These rely on caspase-mediated proteolytic cleavage of a peptide carrying a fluorescent probe. Upon cleavage, the probe becomes highly fluorescent.

NucBlue Live cell stain reagent (Hoechst 33342) is very simple. (As long as you have fluorescent microscope in your department.) Even if you don't have timelaps imaging devices, you just look at cells every few hours. I recommend this because you can get plans of sampling. At the first point, you don't know when cells are going to die, which concentration is good, even if there is apoptosis. If not, you just waste the expensive kit.  Briefly, the picnotic cells can be observed by Hoechst staining. DAPI, you need to fix the cells, and permealized treatment is necessary. Hoechst is membrane permeable. In p53 mutant cell lines, even UV irradiation doesn't cause any apoptosis. Instead, you often observe larger nucleus. It is probably necrosis.   See? two types of cell death can be observed, and you can see when, how, how much cells die from 1 dish. It is my view so I hope this would help you sometimes.

For DAPI staining you do not usually need to fix and permeabilize. It is true that healthy cells will exclude the dye, probably through active efflux. However, during apoptosis, cells are readily stained and nuclear morphology can be assessed. Thus brightly-stained cells tend to be (but not always, beware) dying cells.

Fair enough.  The point is that nuclei staining is cheap, fast and very informative. DAPI is photostable than Hoechst. For immuno fluorescence, DAPI staining is quite easy, 300 nM in wash solution, 5min treatment stains all nucleus. For live staining, I think it needs higher concentration, and particular medium. But it's good when you have already had DAPI in your fridge, please see Justin's reference.

I agree with Sei.

For DAPI, we make a 5mg/mL stock kept at -20. This should be made in water. DAPI is insoluble in PBS, use ddH2O.

Dilute directly into the culture medium, 1:1000. Allow to incubate for about 25 min at 37oC. Visualize under UV epifluorescence.

Thank you all for your suggestions! I tried with TUNEL but I didn't get any signal... I don't know what was wrong in my experiment. I will now try with Annexin V!

Zahia, you could use Acridine Orange / Ethidium Bromide assay. This is a simple, low-cost and effective method for detecting apoptosis. 

You have to prepare a solution containing 4 µg/mL of Acridine Organge and 4 µg/mL of Ethidium Bromide in PBS 1X (its better if you prepare a 100 X stock solution, and then dilute it). 

When you want to measure apoptosis, you have to trypsinise the cells, and when all the cells are detached, add complete DMEM (with 10% serum) to stop trypsin action. Then centrifuge the cells at 1500-2000 rpm for about 2 minutes, then discard the supernatant, and resuspend the cells in PBS 1X, centrifuge again, and resuspend the cells in 100-200 µL of the 4 µg/mL AO/EtBr solution. Quickly, put 15-20 µL in a slide and add a coverslip, then watch the cells in 4-10X fluorescent microscope with UV

Live cells will be green, early apoptotic cells will be also green and with marginalized and brighter green nucleous (chromatin condensation), late apoptotic cells will appear orange with orange core and necrotic will be completely red.

Hope this works for you!

Also, treat your cells with an agent that induces apoptosis as a positive control.

Staurosporine is useful. This way you will know that your assay is working.