Hey,
1) I am performing MTT assays and was wondering how much the cell size can influence the mount of metabolized MTT?
I thought that the amount of mitochondrial membrane, (so also the amount of active enzymes that metabolise MTT) correlates with cell size and therefore lets say 100 normal sized cells could metabolise approx. the same amount of MTT than 160 smaller cells, right?!
2) does anybody know if and how the metabolism of nutrients (Glucose, aa ) is connected to the activity of the MTT metabolizing enzymes ?
As maybe the presence of nutrients at the time point of the addition of MTT !!! can influence the capacity of metabolizing the chemical ..... (I mean : 100 cells consumed a lot glucose --> remaining glucose in medium is less and metabolism goes down...so also the metabolism of MTT, ..... smaller signal,.... but treated cells, where only 50 are present consumed less Glu, therefore still more in the medium when adding MTT and therefore 50 cells can metabolize more than 50% of the MTT than 100 untreted cells)
Thanks a lot for reading and trying to understand and helping :) !
1) To test if your mitochondrial number has increased proportionally you can try Western blotting the lysate from equal number of cells with antibodies against prohibitin or another mitochondrial marker. Alternatively IFA or flow cytometery can quantify fluorescent intensities vs size of cells, again staining with mitochondrial markers.
2) I understand your thought process but I can't help you with the specific metabolics. I will recommend that you use sufficient media for the number of cells, and possibly change the media prior to reading if your cells have been growing for too long. Also, you can try testing spent media vs fresh media using the same cell line and number of cells. If you see a reduction in your spend media than you should have your answer.
Hope that helps. Good Luck.
Thanks, concerning the second part I got an idea.
The first part ... I hoped someone working with Mitochondria could help me,... if generally the membran quantity is proportial to cell size.... and if the catalizing enzymes than are proportionally to the membran ...... otherwise its too complex and time consuming runninng first these experiments, thanks though !
Its not only mitos and succinate-DH, but also ER enzymes depending on NAD(P)H which reduce the MTT - so I would say yes, the larger the cells, the more of these enzymes shoiuld be there - if you are using the assay as a ROUGH estimate of cell viability, whiich you should do.
If you need to know more precise, then indeed you should check if the cells you compare have different amounts of mitos and ER per cell by WB - or at least check that ccell size is pretty similar. Good luck !
For some reasons I do not see why mitochondria number should correlate with cell volume. I think metabolic state of the cell is more important. Another issue here is that utilization of MTT is not purely mitochondria related. I can add to what Rudolf J Wiesner said that it is may be Ok for many oxidoreductases (i.e. alcohol oxidoreductase) to use MTT as an electron acceptor. In fact some researchers argue that only up to 50% of MTT is reduced by mitochondria (see http://onlinelibrary.wiley.com/doi/10.1002/cyto.10080/pdf).
The attached article could (maybe) be of help (if not too late).
Best regards
Robert
what is the interpreation of increaseing of decreasing metabolic activty?
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