Hi Caspase glo works well. I have used it before and was detecting even the small % of apoptotic cells

Hi, I used the caspase glo from promega almost 4 yrs back.It is in 96 well plate format. I remember adding equal vol of reagent and shaking the plate for 5 min before reading the lumi. Follow the kit instructions closely, it is very straight forward.

Thank you very much for your answers!

Hi, Sa! I used the Caspase 3/7, 8 and 9 from Caspase Glo kits and worked pretty well. If you have execution doubts please let me know.

Good Luck!

I have one more question concerning this assay.

I do not want to use 96 well plates. We found that, due to evaporation, there is more medium in the inner wells as compared to the outer wells when the plate is incubated for several days.

Therefore, I usually use 12 well plates or 24 well plates. May I perform the assay in 24 well plates without increasing the amount of Caspase3/7 reagent?

When I use 24 well plates, cells are cultured in 500 μl medium. Before adding the caspase reagent, I have planned to transfer 400 μl of the culture medium to small tubes, centrifuge at 200 g for 5 minutes and resuspend the pellet using the remaining 100 μl. Then, the 100 μl are transferred back to the 24-well plate and 100 μl of the Caspase 3/7 reagent is added.

By this method, I will not loose any detached cells by removing 400 μl of the medium.

Dear Sa,

IWhat i used to do, was remove the medium from the 96 well plate and add fresh 100 ul medium with a multi channel, just before adding the reagent. As far as i remember, you must add equal volume of the reagent to the medium. otherwise the reagent will get too diluted. make sure you include a +ve control and try different time points. This assay is very sensitive and it detects early onset, way before we observe nuclear fragmentation. I got highest signal 12 h after incubation. I used staurosporine as +ve control.

and one more thing. the detached cells didnt give me any significant reading when I tried, because they are the necrotized cells which lost caspase activity. Too many cells may saturate the signal, careful with your seeding density.

thank you for your answers.

Right now, I am thinking about when I should measure Caspase3/7 activity.

I will transfect my cells with microRNA mimics. Two days after transfection cells are stimulated with Doxorubicin (DXR). As I do not even know whether caspase 3/7 are activated in my cells upon DXR treatment, I have planned to analyze after several days.

Day 1: Transfection

Day 2: -

Day 3: treatment with DXR; analysis of Caspase3/7 activity in transfected cells (I want to analyze whether my miRNA alone induces apoptosis)

Day 4: analysis of Caspase3/7 activity (only untreated cells to analyze whether my miRNA alone induces apoptosis)

Day 5: Caspase 3/7 activity of transfected treated/untreated cells

Day 6: -

Day 7:Caspase 3/7 activity of transfected treated/untreated cells

Day 8: Caspase 3/7 activity of transfected treated/untreated cells

thats a good plan. you may want to assay just before adding dxr, to show that your cells are healthy and 12-18 h after dxr addition. I would check the last assay at day 5 because apoptosis is an early event, you will have to deal with a lot of variables when you incubate for such long perio .

All the best :-)

Hi Reema,

0.1 million is what I used/well ( varies for different cell types as the doubling time is different).

I tested nanoparticles at different time points. After addition of np, i checked 12 -13 hrs, 18 hrs, 24h and 48 hrs. I got a positive signal only at 12-13 hrs after addition.

Yes you must substract your blank from the test wells.

And also include a +ve control to make sure the kit is working well.

longer incubation is not reommented as your cells may become confluent and die eventually.

If you see same reading in all wells that means you cell number is too high and thus the signal is saturated.

you are welcome.

store at -20.

For cell culture and annexin, opt time point is 24 h.But for this kit before 24 h would be ideal. This is because it is detecting the early event just after casp 3 is activated. annexin kit detects apoptosis after the activated casp 3 cleaves its substrates.By the time annexin detections comes on casp3 would have done the job.

compare with your positive control and negative control and do a statistical significance if possible

I have one more question. My miRNA is a tumor suppressor miRNA. Therefore, cell viability and cell proliferation decreases after transfection. Therefore, I would have less cells in wells transfected with miRNA mimics as compared to wells in which cells were transfected with NC (negative control)-mimics. The number of cells does not differ so much that you see a difference under the microscope but xCELLigence indicate that cell proliferation significantly decreases.

What about the fact that I do not have the same amount of cells in each well?

From your initial question, what you want to determine is whether DXR kills your cells by apoptosis and whether apoptosis is dependent or independent of caspases. you can approach this problem in a much easier and cheap way using cell permeable caspase inhibitors extensively described in literature. Also, to avoid evaporation in 96 well plates, you should fill up with medium alone the outer wells and only seed cells in the inner wells. This will reduce the useful wells to 60, but it will also reduce the cost of the assay.

Hi Sa,

If you think cell number varies significantly between your treated and untreated sample, you may want to go for annexin-pi staining instead of casp-glo. In annexin-pi you can choose the number of cells you wish to analyse.

Or you will have to count the number of cells in the platesafter treatment and use same number of cells before adding the glo reagent.

Hello Sa,

this is a quite expensive assay and I would only use it if:

1. I was directly comparing several parameters in a quantitative way - e.g. I treat a cell line w/ different drugs and want to compare caspase activation. accurately.

2. I have a lot of parameters to compare and/or small amounts of cells.

In other situations like a qualitative question like "does my miRNA induce caspase cleavage?" I'*d go for Western blot for example. That is if you are producing the miRNA yourself and have enough at your disposal to transfect larger amounts of cells.

If, for the reasons mentioned above, or others you "have to" use casp glo you should measure 12-24h after incubation (drug/siRNA etc.) - and only if you have very slow growing cells (like 5 or more days doubling time) it might make sense to check after 48h. But as you mentioned above, if your controls keep on growing and if they have rather high levels of endogenous apoptosis/casp cleavage your collecting artifacts.

If money is not an issue, promega also offers a combined proliferation/caspase assay which, I think, is even more expensive than casp glo.

@Reem A.m: You don't need to seed the cells into white plates, you may as well at the end of your experiment add the casp glo, shake for 5min and transfer the whole thing into white plates. This is esp. recommended if you're growing adherent cells, so you can monitor them with the microscope before the end of the exp. also your cell numbner very much depends on the cells your working with. We use everything between 10,000 and 40,000 adherent cells/well for a 24h exp.

Also we have ver good experience with at the end of the exp. sucking off all media, diluting the casp glo 1+1 with PBS and add that mixture to the cells. shake 5min, rest 10min, transfer to white plate. measure within the next 30min.

We usually buy the 100ml package, dissolve, aliquot and store at -80°C, which keeps the assay fresh for months/years.

just to add to this: there are cheap enough dyes that can replace PI for the DOXO setting. E.g. sytox blue and the likes. easy enough for use on 96 wells plates. for conditions with high cell death, check with caspase inhibitor-cotreatment if death goes down (e.g. zVAD-fmk or qVD-OPH).

I have used Caspase Glo 3/7 several times and I think it works fine. You can read more about alternative ways to measure apoptosis in Christenson, K., et al. Analyzing cell death events in cultured leukocytes, In Methods in Molecular Biology - Leukocyes; 2012;844:65-86.

I use the Promega Caspase-GLO kits for years now and they work very well. Other alternatives for analysis of Caspase 3 are the Homogeneous caspase Assay (Roche) or

the CaspaTag kits from Chemicon/Millipore.

Yes 30 min is enough

I use the immunochemistry FLICA Assays for many years...good product and good service:

http://www.immunochemistry.com/

Thank you all for your answers. I do not know what I did wrong but the Caspase assay is not working. I spoke with the technical service and sent them my results. However, they found nothing that I did wrong.

Thank you Christian for your answer- I may try the FLICA Assay!

if you need any info let me know! or I will contact you with the support person at ICT that can give you all the information.

Hi reem, caspase 3/7 is basically targeting apoptosis means that if caspase 3/7 are there it undergoes apoptosis so you should have value for it but you also need to remember if in case it does not undergoes apoptosis it might as well contain pro-caspase 3/7 which lead to necrosis which you will be obtaining 0 value. 

Dear Sa, I also used this Caspase Glo kit (for caspases 3/7, 8 and 9) and worked pretty well, with reproducible results. I basically followed the fabricant instructions and in my case I saw caspases 3/7 activation starting early 4 hours after my treatment. If the compound you use to induce cell death is strong, you will need to find the best time point to measure it. Unfortunatelly each kit can be used just once in a 96 well plate (because you cannot thaw and freeze again). Because that you will probably need use more than once. In the first time you standardize treatment time, cell density, drug concentration with a few wells (3 to 5) per condition. And then you repeat with the final conditions. I used 10 to 12 wells per treatment. As a luminescent test, you will need a special 96 well plate, glass bottom, white wall. I have just one of those where I usually transfer the media prior reading. After that just wash to use again. In this way, you can cultivate your cells in a commom 96-well plate. But you mentioned the death you may see is caspase independent, was that right? Than this is not the best assay for you, maybe start with some viability assay. To be sure your test worked, add some positive control (I used 1uM Staurosporine). As viability assay, I suggest the resazurin salt test, it is an Alamar Blue based assay. It is sensitive and is fluorescence based. You might check if the fluorescence of your treatment interferes here. But you can also wash your treatment before the test. Additionally, if you are looking for a test to publish that your compound induces death, you will need at least two different assays to prove that. The assay will vary in accordance which type of death is occurring. I wish good luck and please let me know if I can be more helpful!

Hi all. Following up on this, Promega recommends to absolutely avoid freeze and thawing the reconstituted reagent but I see here people freeze it in aliquots. Do you see any loss of  signal. The protocol says "refrozen reagent stored at –20°C for 4 weeks will give a signal approximately 60% of that of freshly prepared reagent" so I was wondering what happens with reagent frozen for months as someone has said.

Hi Carmela,

Yes we (and all the other labs I know working with this and other luminiscent Promega assays (like cell titer glo)) freeze the stuff for months at -80°C. There may be an absolute loss of signal, I never checked, (although I doubt that it will be 60%) but this will only be relevant if you're comparing raw-values obtained from a fresh aliquot with those from one that has been frozen for some time, running the same experiment.

However, what you'd usually do is run internal controls (i.e. un-/solvent-treated cells and optional staurosporin or another potent killer) and compare caspase activation to that of cells treated w/ your specific drug/treatment. I.e. relative comparisons, which will then (and I tested this) yield a similar result.

Bottom-line is, the company wants you to buy small aliquots which are even more expensive, and use them freshly. There's no harm in that. Except for your funds...

Hello Guys. thank you for the information.

I am using promega Cas3/7 activity kit and it works perfectly well as i compared the level of activation using the kit with WB results of cleaved cas 3. However, i am using the kit to assess the apoptosis after drug treatment using IC50 concentration. My question is: Do we need to normalize the signal to cell number? according to the kit, the signal is linear with cell number i.e. using IC50 means the wells with drug treatment contain 50% cells comparing to the control well. is it enough just to multiply the signal from drug treated wells by 2 ?

Hi Guys,

has anyone ever tried this assay with black plates with clear bottom?

Thank you

Dear Siti,

it will work for sure! Your absolute luminiscence units will be lower and can not be compare to results with other plates but relative results on your plates should be fine.

Dear Abdullateef,

short answer is: No!

just seed equal numbers of cells and measure after short drug exposure. Timing is important here. You measure IC50 e.g. after 3days and want to know WHY there's only 50% left. Is it apoptosis, induced earlier (e.g. after 12 or 24hrs) leading to cell death, i.e. less cells in the well? Casp Glo will give you the answer. Of course it may be mixed with growth arrest. Add other assays to clarify.

Hope that helps.

Reem A.m I found this article, I hope it's useful for why did you got 0 on your measurements

Thomas Mühlenberg you seem like a specialist when it comes to this assay; I appreciate all of your insight and answers to the prior questions. I have another one for you (though I believe I know the answer to it).

We performed this assay over 72 hours of treatments. Contrary to our prior growth/BrdU assays, we saw a decreased luminescence signal with increasing treatment dosages (formerly, our compound decreased cell growth and proliferation in a dose-dependent manner).

Did we assay too late in the apoptotic process? Should we dial back our treatment window to 24, even 18 hours? Of course this is concentration, cell line, and compound specific but I thought that you might be able to give me some suggestions given the breadth of your responses to prior questions.

Thanks,

Zach Bomstein

Hi Zachary Bomstein ,

Seems like the right idea to me. With incresed dose, more cells might die faster and, as they say, dead cells don't glo...

Good luck!

Thomas Mühlenberg thanks for your answer and sorry for the delayed response. We got biologically significant results using the assay after 12 hours of treatment. We also assayed after 24 and 36 hours though, and found something interesting occurring. The luminescence of the control cells in our 24 and 36 hours reads was unexpectedly large; in fact, it was equal to the signal given off by our highest doses at 24 hours, and almost 70% larger at 36 hours. Even more perplexing was that these are all cells seeded from the same line and passage. Does endogenous caspase activity occur with growing cells? Otherwise, I am having trouble trying to square these results with the ones I observed at the 12 hour timepoint.

Hi,

I feel like we're leaving my field of expertise here, so I'd suggest opening a new Question thread, so apoptosis experts may be better able to help.

Just some things that come to mind:

1. Cell density may play a role (--> repeat with more and less cells)

2. Cell line "base line apoptosis" - some show quite high levels.

3. Confirrm your results with another apop. assay, like annexin V/7AAD by FACS, or caspase by western, etc. (keep 1. and 2. in mind)

Hope that helps!