Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon

Round 1

Reviewer 1 Report

Revisiting the resazurin-based sensing of cellular viability: widening the application horizon

Darja Lavogina, Helen Lust, Maris-Johanna Tahk, Tõnis Laasfeld, Hans Vellama, Naila Nasirova, Markus Vardja, Kattri-Liis Eskla, Andres Salumets, Ago Rinken and Jana Jaal

Herewith I am submitting my reviewer comments for the above mentioned manuscript, which is under consideration to be published in Biosensors. The article is about NAD(P)H-aided conversion of resazurin to fluorescent resorufin which is used to evaluate cell viability. Generally, there are many viability/metabolic activity tests that are not used in a standardized way and optimizing the methods is an important task to do in order to produce comparable data. Thus, I found the topic worth investigating.

Overall, the article is well written and clear and the level of English is satisfactory (as far as I can judge) The figures are prepared well to aid understanding (for instance the color code of Figure 2 is very nicely selected). I found that the paper was done very thoroughly. I only have some very minor comments:

Page 14 Figure 7: The area that was observed is given as fields of views per well. But I would rather convert it is some more standard units (Like um2, mm2 or whatever is convenient). Without reading further in the text it is not clear what the field of view is here in this context.

Author Response

biosensors-1635664

Response to reviews

We thank the reviewers for their valuable comments. Below we provide the point-by-point response to the raised issues.

Reviewer 1:

Page 14 Figure 7: The area that was observed is given as fields of views per well. But I would rather convert it is some more standard units (Like um2, mm2 or whatever is convenient). Without reading further in the text it is not clear what the field of view is here in this context.

Authors:

• We introduced the requested changes to the Figure 7 as well as the Supplementary Figure S9. We also modified accordingly the legends of the corresponding Figures.

Reviewer 2 Report

The study of cell viability is a key, very common research method in cell biology, pharmacology, interdisciplinary research, etc. Various approaches are used to determine viability and functional activity, including those based on dyes that change their spectral characteristics in response to changes in cell viability. The ideal test forin vitrocell proliferation and cytotoxicity is a simple,   rapid,   efficient,   reliable,   sensitive,   safe   and   cost-effective  measurement  of  cell  viability.

The authors analyzed in detail and qualitatively the application of the resazurin-based sensing of cellular viability. However, there are some questions and remarks:

For control, the authors took methods for determining toxicity - SYTOX Blue assay and Casper3-GR. Choice not well understood. The SYTOX Blue assay is used to quantify dead cells by flow cytometry. Since the % of living cells is calculated in relation to the total number of cells. The intensity of the nucleus stained with this dye can be different at different stages of apoptosis. With hypercondensation of the nucleus, the fluorescence is brighter, which can introduce errors into the analysis. The Resazurin assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion is intracellular, facilitated by mitochondrial, microsomal and cytosolic oxidoreductases. A more suitable comparative method is MTT. Moreover, the text provides a theoretical comparison of these methods (lines 284-289), but in practice it has not been applied.

Since the article is methodological, in conclusion, I would like to see specific recommendations for the use of the dye, which the authors identified as a result of such an extensive study

Author Response

biosensors-1635664

Response to reviews

We thank the reviewers for their valuable comments. Below we provide the point-by-point response to the raised issues.

Reviewer 2:

For control, the authors took methods for determining toxicity - SYTOX Blue assay and Casper3-GR. Choice not well understood. The SYTOX Blue assay is used to quantify dead cells by flow cytometry. Since the % of living cells is calculated in relation to the total number of cells. The intensity of the nucleus stained with this dye can be different at different stages of apoptosis. With hypercondensation of the nucleus, the fluorescence is brighter, which can introduce errors into the analysis. The Resazurin assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion is intracellular, facilitated by mitochondrial, microsomal and cytosolic oxidoreductases. A more suitable comparative method is MTT. Moreover, the text provides a theoretical comparison of these methods (lines 284-289), but in practice it has not been applied.

Since the article is methodological, in conclusion, I would like to see specific recommendations for the use of the dye, which the authors identified as a result of such an extensive study.

Authors:

• We would like to emphasize that the SYTOX Blue assay as well as the Casper3-GR assay were not used as controls – our goal was to show that the resazurin assay can be used in combination with those in case if users are interested in more thorough examination of the cell death process. We phrased this consideration correspondingly throughout the text (g., line 29 “possibilities for combining the resazurin assay with other methods”, lines 70-71 “address the risks related to combination of resazurin-based sensing with other fluorescent molecules”, Section 3.7 “Combination of resazurin assay with assays utilizing Casper3-GR biosensor and SYTOX Blue dye” – the mentioned line numbers are valid for the Tracked Changes version). Still, we fully agree that the manuscript would benefit from comparison of resazurin and MTT assays. Therefore, we introduced the following changes into the main text and Supplementary data:
  • To the Supplementary data, we added a table comparing the technical characteristics of either assay (Table S3).
  • We carried out two pilot experiments where we characterized the dynamic range and the performance of either assay in four different cell lines. The experiments are described under the new Section 2.6 and the results are summarized in the Supplementary Figures S16-S17.
  • We added Section 3.8 to the main text of the manuscript where we provided cross-references to the new data listed above and discussed the technical feasibility of either assay.
• We added two example protocols to the Supplementary data that provide initial instructions for the potential users who would like to utilize the resazurin assay. Also, we modified the concluding paragraph of the main text (lines 881-885 in the Tracked Changes version) by adding cross-references to the Supplementary Table S2 (listing the suggested cell seeding densities) and the Supplementary protocols.

Round 2

Reviewer 2 Report

The changes made have improved the quality of work