I have used an Ac-DEVD-AFC substrate before, instead of AMC, but results should be similar. The buffer I used contained 100 mM HEPES (pH 7.25) with 10% sucrose, 0.1% NP-40 and 10mM DTT. Final concentration of AFC was 50 uM. We incubated for 2 hours at 37C. I hope this might help!

Thank you, Gabi. Which wavelengths for Ex/Em did you used?

We used Ex.: 400nm, Em.: 505nm

I have checked our reference years ago ( Zhang et al., Int. J. Cancer: 113, 397–405 2005), and found that the contraction of NP40 we used was much lower (  10 ^-6 % NP-40); our reaction buffer contains 0.1% 3-[(3-cholamidopropyl) dimethylammmonio] propane-1-sul-phonic acid (CHAPS), and the final pH was 7.25.  DEVD-AMC was 50 uM and the reaction was monitored  in a Fluoroskan II plate reader. Hope the answer could be helpful for you.

Thank you very much. I will modify my protocol accordingly.

Dear Lara: I do not have experience with the Ac-DEVD-AMC substrate. However, to detect caspase-3 activation, I do use NucView™ 488 Caspase-3 substrate (Biotium; https://biotium.com/product/nucviewtm-488-caspase-3-assay-kit-for-live-cells/), which it doesn't require a special buffer. This substrate works perfectly with culture media; no fixation neither permeabilization is needed, due that is a live-cell assay. For more details, check the following reference: Cell Oncol (Dordr). 2016 Jun;39(3):265-77. doi: 10.1007/s13402-016-0272-x.  You can use flow cytometer or fluorescence microscope platform to quantify the number of cells exhibiting caspase-3 activation. This is just in case that you do decide to purchase a new kit. Good luck! With kind regards.

Thank you for your suggestions.

Dear Lara

I too would like do dosage caspase 3, however in skeletal muscle (soleus and gastrocnemius) samples. I found a NIH protocol that using DEVD-AMC substrate, to it. But this protocol require that I use 50uM of DEVD-AMC substate with final concentration in buffer assay. However, I don't found with make a standard courve, because I don't found the molecular weight (g/mol) of substrate. So I ask you: how you dilued the substrate and make your standards concentrations?

Regards,

Éder

Dear Eder,

standard curve is made with 100 nM, 500 nM, 1, 2, 4 and 6 uM substrate concentrations. MW of Ac-DEVD-AMC (Cayman chemicals) is 675.6, so you can calculate required concentrations.

Kindly,

Lara

Lara

Thank you so much for you reply. Your comments will be great of value.

Hug,

Éder

Dear Lara,

This is not a answer, more like a question. I currently have difficulty with Ac-DEVD-AMC assay and I wonder if yours has finally worked out.

We purchased this substrate (Ac-DEVD-AMC) and tried to use it to measure caspase 3/7 activity. However, the fluorescence reading from apoptotic cells (staurosporine treated) and control cells are very similar, and about twice of the substrate only conditions. I tried to optimize buffer conditions too because I thought maybe the enzyme is deactivated somehow, but nothing has worked out.

Did you have any luck with this assay? Your input would be valuable. Thanks.

Best,

Nasi