What cell line are you using? I work with neurons, and have used DAPI and Caspase-3 staining to identify pyknotic neurons. I would identify dying cells when I would see very condensed or blebbing nuclei. I have looked at your photographs, and some cells seem to show what appears nuclear blebbing. I would confirm with an experienced eye, and I would practice at becoming proficient at identifying characteristics of apoptotic cells. Best of luck with your work.

What cell line are you using? I work with neurons, and have used DAPI and Caspase-3 staining to identify pyknotic neurons. I would identify dying cells when I would see very condensed or blebbing nuclei. I have looked at your photographs, and some cells seem to show what appears nuclear blebbing. I would confirm with an experienced eye, and I would practice at becoming proficient at identifying characteristics of apoptotic cells. Best of luck with your work.

Alicia Barrientos, i am using colon cancer cells line HCT-15 and HCT-116.

Cells undergoing apoptosis will have nuclear fragmentation, but before that you should see nuclear blebbing, where small parts of the nucleus bud off. However, your pictures show very little sign of blebbing or fragmentation. Have you measured viability (MTT or alamar blue assay) or stained for annexin V to check if the cells are indeed dying?

You have to measure cell viablity and try to use TUNNELL asay to prove apopotosis. DAPI is only indirect marker of apoptosis. And you should alos separate apoptosis and necrosis. Please, combine DAPI with TUNNELL, it will shown you statistics how much cell may undergo apoptosis, 

Hi, I would also have doubts, wether relying solely on morphological parameters in DAPI stainings (nucleus pyknosis, condensation and fragmentation) may not be misleading in some cases, especially for an untrained observer. I personally combined DAPI stainings with caspase3/7 stains to look for colocalized signals. Doing this you can easily calculate the ratio of apoptotic cells (for which casp3 is a reliable marker) vs. total cells. You can even automate the counting procedure without expensive software: ImageJ is freely available and can be equipped with very straightforward cell counting tools!

Hi, I can hardly see any sign of bubbling nuclei, a step which should precede nuclear fragmentation. According to my experience, the nuclear fragmentation is a late event during the apoptotic process, so first you have to be sure that your cells are dying and have some information,s about the kinetic of the cell death with you cells and your experimental conditions. Did you mesure IP staining? (or annexin V), capsase activity, mitochondrial potential ?

Hello Guys, nice discussion going on!

Salman, I would also suggest to use some positive control like doxorubicin so that you can compare the morphology of cells treated with your compound to the morphology of the cells in which you induce apoptosis with positive control. Quite easy to compare!

Hope this helps!

Regards,

Abhijit

Totally agree! However I have to say that from your images I could hardly say that there is any ongoing apoptosis there.

You could try a simple viability assay such as methyl green (see enclosed publication), although it may underestimate early apoptotic events (but probably as much as many of the forementioned techniques). You can always try culturing for longer periods. I would also echo Abhijit's suggestion: use a positive control (dxr as he said or any other).

Best of luck,

Daniel

Article A fast, low cost, and highly efficient fluorescent DNA label...

Salman,

we worked on primary murine hepatocytes some time ago judging bile acid induced apoptosis by morphology changes as visualized by DAPI staining.

You can have a look here and compare normal diploic hepatocyte nuclei with condensed apoptotic nuclei (labelled with arrowheads):

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195080/figure/pone-0025942-g005/

It might be easier in cell lines/primary cells than in tissue sections, I have to admit but it worked quite nicely for us.

Good luck.

I agree with the above comments that DAPI staining is not really a good method to detect apoptosis. In old publications you may find people using bleb formation as an indicator for apoptosis but usually it is later stage. TUNEL has been used as an adjunt method but I have been criticized that it is not a good methods either. MTT/XTT assay is usually not for apoptosis but for cell count or cell proliferation and requires adequate mitochondrial function. Other assay, such as caspase-3, need to be used to convince reviewers if you plan to published your findings.

Hi Salman,

Distinguising apoptotic cell death from non-apoptotic cell death is not straight forward,  and it is best not to rely only on a single assay (TUNEL assay can also give false-positive results in some cases of non-apoptotic cell death for example).  For your DAPI staining,you would expect to see smaller and more brightly stained nuclei if the cells are dying (or have died) by apoptosis (apoptotic chromatin condensation intensifies the DAPI staining). We commonly use Hoechst in stead of DAPI to visualize this. I am not familiar with the cells you are using, but a positive control like doxorubicin would be very usefull, as well as a negative control of untreated cells.

Preferably you combine visual assays (such as your DAPI) with biochemical assays (caspase activtaion, can be immunostaining of fixed cells or western blot on lysates).

Our lab discussed several methods you can use and we also made a flow-chart on how to proceed if you want to identify the type of cell death induced by a certain trigger/compound (in attachment). You can acces the full text on the ResearchGate page of Tom Vanden Berghe or myself.

Just a note of caution: we have noticed that some compounds can trigger apoptosis in one cell line and necrosis in other cell lines.

Good luck with your experiments!

Hello 

I would not use DAPI as your main marker to gauge apoptosis, but rather as a rough guide.

Interestingly, the early phenotype of cell division looks similar to early apoptosis, so beware. I use Hochest rather than DAPI for nuclear staining, which should be similar. According to my personal experience, none (or at most only one) of cells in your photo were in apoptosis. I found apoptotic cells (late phase) have a seperated nuclei composed by clusters of blue dots (apoptotic bodyes?) and the nuclei was smaller than that in normal condition.

By the way, I think apoptotic body based on nuclear staining was not good for quantitative anaylsis of cells apoptosis. Cleaved-caspase would be better. If you are interested in early phase apoptosis, you could try dye for mitochondrial membrane potential or Annexin-V-FITC.

May it help you and good luck.

 Hello, Zhanguang Zuo!

From the images, it is clear that the DAPI has stained nucleus, but it is looking that:

1) Either you have used very high conc. of DAPI solution.

2) Incubation time is very long.

3) You didn't do proper washing after staining (if you are not using DAPI with mounting medium).

I recommend "VECTASHIELD Mounting Medium For Fluorescence With DAPI....... H-1200......Vector Laboratories, Inc.". 

This solution is ready to use. If you are using other dyes also, use DAPI in the last. Just fully cover your cells/tissues with this solution and put cover slip. Wait for 5-10 minutes, fix the cover slip on the slide with some glue, and take images with fluorescence/confocal microscope.

I hope it will work for you.

 I found many TUNEL positive nuclei or fragment of nuclei are NOT stained for DAPI. So DAPI is not a useful marker of dying cells at all.