I would like to monitor cell metabolism over a period of several days (possibly beyond a week). Which of the two methods is correct? Or are both okay to do?
a. Make separate wells for each end-point time (e.g. after 11d, after 3d, etc.)?
b. Use the same well at each time point?
Thank you very much for clarifying
Dear Christine
I know it would be convenient to have the same culture (well) for each time point, but it turns out that Alamar Blue are actually reduced from resazurin (blue, weakly fluorescent) to resorufin (pink, highly fluorescent) which is further reduced to hydroresorufin (non-coloured, nonfluorescent) (refer to Eur J Biochem. 2000 267:5421-5426). So if you use Alamar blue as metabolism indicator you will have to add it at the different time points (60 minutes before you measure the fluorescence). This means that you will have to make separate cultures (wells) for each end point. We however, were able to monitor metabolism for short periods 2-4 hours in one culture, but you must consider that resofurin accumulates in the media leading to overestimation of the redox state of the culture. I would include a secondary analysis such as luciferase assay (ATP) for comparison, as well as directly looking at [metabolite] such as [lactate] (and/or ethanol if your cells produce it), [glucose] and NADH/NAD+.
Good luck with your experiments!
Regards
Marina
Dear Marina,
Thank you so much for the helpful answer. That is indeed what I have been doing, but I was worried if it's valid or not since I'm not using just 1 plate to monitor over the entire time. Thank you so much for the informative answer.
Dear Kevin,
Thank you very much for the information. My only concern is that I monitor over a week, and I'm not sure if this new product will be amenable to that? But I will check the product nevertheless. Thank you.
Best Regards,
Christine
Dear Michael,
Thank you so much for the practical advice. May I just clarify, so if I reuse the well, for example I'm culturing cells in it,
then at the first endpoint, I change the medium to 10:1 med:ab, incubate for 4h, then measure absorbance
then I replace it with medium again and continue to culture
then at the 2nd endpoint, I repeat what I did at the first endpoint.
Is this correct? Thank you so much.
Christine
Hi all,
I have conducted few experiments to see how Alamar Blue reduction varies over a period of 72 hours for different cell densities of a cell type of interest. I incubated cell densities (cells/mm^2 surface growth area in a 96 well plate) of 50, 100, and 200. I added 10% alamar blue and kept monitoring the reduction every 12 hours for 72 hours. Is this wrong? I thought alamar blue can be used to keep monitoring the cells for an extended period. Please give your insights on this.
Dear Ishan
The problem is hydroresorufin that you will not be able to measure. However, the viable/usable measurement time will depend on the rate of resazurin formation, and therefore on the number of cells respiring. Have look at the following article: Eur J Biochem. 2000 267:5421-5426.
Regards
Marina
Thanks both. Marina, that paper was really good! Appreciate your technical input on this.
Hey, unfortunately my post is not much of a solution for your question but merely a new one.
From reading the comments, I can add alamar blue to my 24 hours 96 wells with cells, read the fluro/abs and then remove all media/alamar and add new media, allowing me to then perform the same the next day for 48hours test?
Currently making a plate for each reading
Thanks, great information added here
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