It may sound stupid, but, did you pick up the medium at the time of collection of cells? You must pick up the medium of the cells, since these are the cells that are dying. I mean, obviously, but I've seen people trying to see death in living cells.

It may sound stupid, but, did you pick up the medium at the time of collection of cells? You must pick up the medium of the cells, since these are the cells that are dying. I mean, obviously, but I've seen people trying to see death in living cells.

Maybe 24 hours of treatment with CoCl2 are not enough, did you perhaps try 48h?

Are you sure that Co is an apoptosis inducer?

Have you seen any cell death after treatment with Co? In many case Co does not induce any apoptosis. You have to add Co in the pre-cultured cells and without changes of the medium, You have to use more effective treatments like menadione, which is more direct apoptosis inducer. Good luck!

Cleaved Caspase 8 need more protein loading if Co did induce the apoptosis on your cells. In my experiments, I loaded 40ug/lane (at least) and ran 15% SDS PAGE to detect the Cleaved caspase proteins. And also you should considerate other possibilities in above mentioned.

Maybe you should test other well-known inductors of cell apoptosis such as camptothecin, which also activates caspase-8 in many cell models. See some examples attached...

Another...

Dear Yuning,

I think you should first make sure that COCl2 is really inducing apoptosis in your system.

Perhaps, as already suggested by Dr. Vidal, COCl2 at the concentration you use or after 24h incubation does not trigger apoptosis in your cells. Thus, I would suggest to double or triple the concentration of COCl2 in the medium and incubate up to 48 hours to see whether you get apoptosis. In addition, I would suggest using the Caspase 3 (cat. n. 9662) or Cleaved Caspase 3 (cat. n. 9661) antibodies from Cell Signaling Technology to detect apoptosis associated with TUNEL staining. These antibodies work well in our hands for western blot analysis. As a positive control, I would suggest using the same cell line(s) you are using treated with 1 μM Staurosporine (a strong inducer of apoptosis in vitro). I hope this helps!

Some cell lines have different sensitivity to the apoptosis inducers. You can use camptothecin (up to 10 ug/mL, 24-48 hours, 20 000 cells/well) as said by Santiago Roura. After that, you can do a proliferation assay (MTS, MTT etc..) to compare with untreated cells, in order to evaluate if yours cells are dying or not (you can see that under microscope as well). If so, you can do the western bloth. If you need further proof of apoptotic DNA, recover the medium, purify the DNA and load it on agarose gel (you will see the typical DNA ladder, which is the hallmark of apoptotic cells).

Good luck!!

Dear Francesco,

we usually analyse induction of apoptosis by using flow cytometry following analysis of both Annexin V expression and 7-AAD staining. However, I believe that your advice to visualise DNA damage on agarose gel seems really reasonable and interesting. Many thanks...

Thanks Santiago!! I know Annexin V expression analysis, but I don't trust only for one technique :)

Dear Francesco,

I also believe you gave great suggestions! As you said, the microscope is the first and quicker instrument to see if there is cell death. Of course without cell death at the microscope is highly unlikely that there is apoptosis in the experimental system.

Hi everyone,

I was following the apoptosis induction protocol from this attached paper. I stained my cells treated with 200uM CoCl2 (treated for 24 hours) using Quick Diff staining solution, but I didn't see any apoptotic cells.

Original paper

Dear Yuning,

Do you see any cell death at the microscope? Could you please send me some pictures of the cultured cells under CoCl2 treatment?

Thanks guys, this discussion is becoming very intersting! Don't stop us now...

Hi, Yuning, in the paper you cite there is not too much details about CoCl2 treatments. But details is very important! I would suggest to first check cell viability after Co treatment in your system with either fluorescent dye or even with Evan Blue. If cells did not start to death after 24 hours, you do not need to do any biochemistry. If you aim is to induction of the apoptosis, just switch to more effective inducer. You can repeat again with CoCl2, of course, but only in the case if your study related with Co as a heavy metals. Good lick!

Some antibodies to caspases simply do not work. You will hardly see decrease in pro-caspase band, detection of cleaved caspase is better option. Unfortunately, levels are usually low and it is hard to get a good signal. You should very carefuly select the antibody. Or try to detect cleavage of PARP (caspase substrate), this is much easier (C2-10 antibody works perfectly).

Hi, we have recently assessed levels of both pro-active and active Caspase-3 in left ventricle samples from patients suffering dilated cardiomyopathy by using two different commercially available antibodies (anti-CPP32 purchased from Santa Cruz Biotech. and anti-active Caspase3 from Abcam Ltd). Although some authors had concluded that apoptosis is a hall marker of myocardial damage in these patients, from our own data just a slight increase in pro-active caspase-3 protein amounts is found in diseased compared to healthy myocardium. Additionally, there is no detectable levels of the active protein form.Thus, in accordance with that previous background, we suggest thatardiomyocytes start apoptotic self-destruction but do not entirely complete it, thus rendering the cell functionally compromised, but still viable, in human end-stage heart failure. In summ, unlike another more clear and widely-accepted indicator as collagen deposition by using circularly polarized ligh after Picrosirius red staining, the exact role and contribution of apoptosis to human heart failure remains under active or conflicting discussion...

Dear Santiago,

You have very interesting data! Have you tried to stain the tissue sections with TUNEL to see whether the damaged cardiomyocytes stain positive or negative?

Yes, you are right. There is a very interesting debate in this field... Of course we proved it but the result was not overall conclusive. Following a bibliographic revision, several limitations exist in relation to some of the most widely-used techniques to measure apoptotic cells such as TUNEL. Briefly, it is unclear how many DNA strand breaks result in a positive signal in the TUNEL method. Due only 10% of all DNA fragmented seems to be required for a positive signal, the level of apoptosis may be highly overestimated or this contribute to falsely levels of cell death (see Schaper et al. Circ Res 1999;85:867-9). Furthermore apoptotic, necrotic, undergoing DNA repair and living cells can be equally detected by TUNEL (see Kanoh et al. Circulation 1999;2757-64), rendering the use of this method even more questionable... We stayed with collagen deposition which was less controversial!

Thank you very much for the kind explanation! I come from a Pathophysiology background and, although I teach heart failure to students, I was not aware that damaged cardiomyocytes were in this status of incomplete apoptosis.

Thanks Diego! This is not, however, applicable for all types of heart failure. Programmed cell death has been involved in a variety of myocardial diseases such as myocardial ischemia-reperfusion (see Gottlieb et al. Ann N Y Acad Sci 1999;874:412-26), ischaemic cardiomyopathy (see Saraste et al. Circulation 1997;95:320-3) or myocarditis (see Kyto et al. Am J Cardiol 2004;94:746-50). Conversely, in DCM, there is an active discussion (see, e.g Schaper et al. Herz 1999;24:219-24, Westphal et al. Mol Med;14:55-63, and Bott-Flügel et al. Eur J Heart Fail 2008;10:129-32). Although some studies have reported putative increases in the levels of several apoptotic markers such as caspase-3 activity, DNA fragmentation (TUNEL) and electron microscopy (see Narula et al. N Engl J Med. 1996;335:1182-9), others have confirmed significance only in patients with decompensated heart failure in which a striking increase in caspase-3, but not DNA fragmentation were observed...

I've done the CoCl2 titration and some pictures were taken. The viability and morphology changed significantly when the concentration of CoCl2 reached 400uM (24h).

control cells 0uM CoCl2

Control DAPI staining

400uM 24hours

400uM 24hours DAPI

Dear Yuning,

There is clearly apoptosis now in you cell culture. This can be seen on DAPI staining since the shape of nuclei is altered and some of them are undergoing degradation. I am sure you will have increase in cleaved Caspae 3 and cleaved PARP levels by western blotting now.