Confusing caspase 3 results (no literature to explain?) - (Apr/09/2007 )
Dear All,
I’m so pleased to have found this forum! 
I’m currently floundering and thought it was only me in this big ol’ science world who has problems! Have already learned a lot by reading through all the posts.
I'm new to the site as you can see, I'm also a novice 'baby' scientist - so please forgive me if my explanations aren't great. But I'm very desperate for some help - so here I am!
I'm currently working on my undergraduate dissertation; I'm evaluating the apoptotic effect of a polyphenolic compund on HT-29 cells. So far, I've established that viability is reduced by increasing concentrations of my polyphenol using an MTS assay. Then, using flow cytometry, I've determined that these same concentrations correlate with increased apoptosis rather than necrosis (using annexin V and propidium iodide) over 8 and 24 hours. All great so far. BUT! I've then gone on to assess caspase-3 activation using the AnaSpec Rh110 Caspase 3/7 assay kit. My results make no sense whatsoever. My live (untreated) cell control has HIGHER caspase-3 activity than my treated cells. Furthermore, as my treatment concentrations increase - caspase activation decreases somewhat. This occurs after both 8 and 24 hours treatment.
Having seen increased apoptosis using flow cytometry - I know that the cells aren't merely necrosed, so this doesn't explain it. Plus, I've found literature that describes quite clearly that the same treatment that I'm using, at the same concetrations, in the same cell-line, induces caspase-3 activity commencing at 8 hours - and increasing continually over 24 hours. So methinks I've gone wrong somewhere.
I've performed both the 8 and 24 hour treatments 3 times - each time in triplicate. The results are always the same - although there seems to be more of a decrease in caspase-3 activity over 24 hours as the treatment concentration increases. My treatment did induce an increase in fluorescence - so I blanked this from my data. I admit that I used McCoys media which contains phenol red - BUT before you all scream out that I shouldn't have, I considered this when planning the experiment. A number of the PhD students working in the lab have used phenol red-based media in this experiment, and found that the results were comparable to non phenol-red. Also, I had to sub-culture my cells into an alternative media recently, and they were really unhappy, so I didn't want to run the risk of confounding my results by changing the media (as this is 'only' an undergrad dissertation - I don't have much time to try different things). And even if the phenol red is having some kind of odd effect - I can't find any reason whatsoever why it would make my results the reverse of what I should see! And why is there more caspase-3 in my live cell control? Finally - I viewed the cells under the light microscope, and they looked as they should. Seemingly more apoptosis at increasing concentrations and some necrosis at the top end. The live cells looked - well, ya know - alive 
I've spent the last day and a half trawling Google, Web of Science, Pubmed (etc, etc) and can't find a single thing to help me explain my results. If anyone has the time and energy to read all this and get back to me with some answers, I'd be hugely grateful. I'm meeting with my supervisor on Wednesday and would love to be able to tell her something that makes sense!
Best wishes,
Sue
have you explored interactions of assay components with reagents or compounds in your samples? I'm not familiar with the caspase assay, but similar problems can occur often in colormetric or fluorometric assays. What kind of indicator is used in your caspase 3 assay?
Thanks for your quick reply. This gives me something else to look at. The indicator is (Z-DEVD)2-Rhodamine 110, which emits a "bright green emission".
The treatment I'm using is the tea polyphenol EGCG - I'll start looking now - but if anyone has any prior knowledge of any interactions between these, I'd be grateful for a heads-up!
Thanks again,
Sue
I notice a few of you have read this topic - so just in case you're interested - I still have no answers!
I've looked at every possible angle that I can think of, and nothing makes sense! Interstingly though, I've learned that a PhD student using the same assay on the same cell line as me, also had bizarre results. Perhaps the cells just don't agree with the assay!
I've looked at every possible angle that I can think of, and nothing makes sense! Interstingly though, I've learned that a PhD student using the same assay on the same cell line as me, also had bizarre results. Perhaps the cells just don't agree with the assay!
I have previously read about caspase 3 being active without inducing apoptosis, but I can never find the papers without knowing exactly where they are. You may want to try using AnnexinV/Propidium Iodide. If cells show AnnexinV on the cell surface, they are guaranteed to die within 24 hours. Also, the manufacturer should be aware of activated caspase 3 not inducing apoptosis, perhaps they could direct you to the references.