Dear Azra,

It is relatively easy to perform such experiments as dynamically illustrated in the 4 attached videos and described / detailed in the attached articles.

With respect to the videos:

1. Hs683 is a human brain tumor cell line and ferrocifene is a CYTOSTATIC compound that induces senescence (thus very few cell deaths; see the attached article by Bruyere - Mathieu et al);

2. SKMEL-28 is a human melanoma cell line and sphaeropsidin A is a CYTOTOXIC compound that induce high levels of cell deaths (see Mathieu et al., 2015).

The other attached articles provide you with detailed description of the methodological approach I propose you to use.

In my very own opinion, you cannot "follow" the "future" of an INDIVIDUAL cell by the MTT assay because of physical limits in terms of sensitivity of the method.

Best regards

Robert

After adding MTT to any cells you will not find any viable cell. All the cells will have Formazan crystals as soon as the MTT interacts with their mitochondria. Only viable cells can do this MTT to Formazan conversion. While looking into the microscope in bright field you should use another counter stain which only stains dead cells. Trypan Blue may help but this will be the same color as formazan crystals. Only differentiation will be the physical appearance of both as one will be like a crystal or needles and other will be smooth.

Good luck!!

Dear Robert Kiss, Thank you so much for providing me with these information. But could you please explain in more detail what you mean by physical limits in terms of sensitivity of the MTT method (in your last sentence) for tracking individual cell's viability?

Thank you Vivek Vaish  for your helpful explanation. But I think cells with Formazan can still be alive until they form needle like crystals. At least this is what my results shows. They can only be considered as dead cells when vesicles consisting Formazan  convert to needle like crystals. What do you think?

Dear Azra,

I mean that I really do not think that the MTT-related signal emitted by one single cell is sufficient in terms of absorbance for a spectrophotemetric analysis.

Best regards

Robert

Dear Robert Kiss,

Thank you once again for the explanation. However, I think I haven't been good enough in describing my experiment. So I should emphasis that my experiment was not to use spectrophotemetric analysis in order to distinguish a single live cell. Instead, I wanted to image the cells using bright field microscopy while MTT is being converted to Formazan crystals in a single live cell. Something similar to what is described in this paper:

https://www.ncbi.nlm.nih.gov/pubmed/9231715

Best regards,

Azra

Dear Azra,

Best way for you to do single cell viability test would be using fluorescence microscope and fluorescent dyes.

But if you still want to use bright field then only trypan blue staining is enough. Else you can find out some other live cell stains with Thermo Scientific or Sigma which would give a contrast staining then Formazan crystals.

Good luck!!

Dear Vivek,

Actually, the main idea is to check whether MTT assay works for cell viability assy in single cell level. I do agree that otherwise it would have been much easier to  fluorescent dyes and use fluorescence microscopy. However, my experiment with MTT worked out and the result were satisfying.

Thank you very much for your time.

Best regards,

Azra