An Optimized Flow Cytometric Method to Demonstrate the Differentiation Stage-Dependent Ca²⁺ Flux Responses of Peripheral Human B Cells

Round 1

Reviewer 1 Report

In the article, the authors provide a very comprehensive description of the basic research on An optimized flow cytometric method to demonstrate the differentiation stage-dependent Ca2+ flux responses of

peripheral human B cells. 

It is evident from the article that the authors have been working on this topic for a long time and therefore did not find it necessary to explain the development of flow cytometric methods for the experiment in the introduction. Instead, they discuss B cells , their pathologies, and other matters unrelated to the experiment. 

- please revise to topic

The structure of the paper is unsatisfactory and does not meet the editorial requirements for papers.

The technical aspect is certainly interesting but cannot be evaluated in the above context.

One last note - the literature has no defined format. In particular, citation 39 is bizarre. 

Author Response

Comments and Suggestions for Authors

In the article, the authors provide a very comprehensive description of the basic research on An optimized flow cytometric method to demonstrate the differentiation stage-dependent Ca2+ flux responses of peripheral human B cells. 

It is evident from the article that the authors have been working on this topic for a long time and therefore did not find it necessary to explain the development of flow cytometric methods for the experiment in the introduction. Instead, they discuss B cells , their pathologies, and other matters unrelated to the experiment. 

We highly appreciate your review, the comments, and helpful suggestions. According to them, we have made the suggested changes and amendments. Please see the corrections below.

- please revise to topic

Thank you for your suggestion, the following sentences were added to the introduction

Several options are available for tracking the changes of the intracellular Ca2+ concentration of cells. Patch clamp methods allow for the precise characterization of Ca2+ flux on a single cell level [30] but do not provide the option to describe entire populations simultaneously. With the development of flow cytometric Ca2+ responsive probes, real-time monitoring of the Ca2+ flux response of selected cell populations became possible. For instance, Fluo-3 and Fluo-4 are both widely used fluorescent Ca2+ indicators that are excited by the 488 nm laser with an emission spectrum in the low 500 nm range, but Fluo-4 is brighter, permeates the cell membrane better, and has a large dynamic range for reporting Ca2+ [31].

The structure of the paper is unsatisfactory and does not meet the editorial requirements for papers. The technical aspect is certainly interesting but cannot be evaluated in the above context.

Thank you for this comment, we have reorganized the ‘Result’ section to improve the flow and make it easier to read. There has been a mistake during the insertion of Figure 2, therefore a previous version was incorrectly inserted, creating confusion. Please see updated and corrected Figure 2 and the corresponding legend. Figure 6 has been renamed Figure 3, consequently Figure 3, 4, and 5 is now Figure 4, 5, and 6 respectively. Figure 3 has been modified to match the flow of the text. The text of the ‘Results’ section have been reorganized to correspond to the order of the Figure everywhere.

 One last note - the literature has no defined format. In particular, citation 39 is bizarre. 

Thank you for this comment, indeed there was a mistake in Endnote during the importing of the format into the template of the journal. Several tags were also lost during the import, this has also been corrected. This has been corrected and the format is now according to the Endnote format provided by the journal. Indeed, this citation (now No. 42) is strange, due to the large number of Authors. We have manually deleted all authors above 10, however, the final formatting is up to the Editors, as this is generated based on the settings of their Endnote template.

Reviewer 2 Report

This paper describes a method that is potential to be used for clinical samples, but it lacks the simple well-defined methodology for routine use. It could be a pioneer for such a work for sure. The method generates a lot of data and the kinetics from different B cell subsets may show different functionalities of the cell type. In general, please consider changes for the following comments.

1. Editorial changes: Ca2+ vs. Ca²⁺ throughout the manuscript.

2. The flow of results does not correspond to the figures. Figure 6 came before Figure 4, and sub-figures came before another in the text, leading to unnecessary confusion, especially for Figure 2.

3. LINE 115, the text talks about NSw memory cells but the Figure links to Naive B cells (Figure 2/A). Is this an error?

4. From LINE 127 to LINE 132. The text does not belong to Figure 2. The Figure 2 description (LINE 133 to 137) has to re-written (C to G). It should be A to E.

5. LINE 147, Is that Figure 2/A? It's about Sw and DN.

6. LINE 151-153, there is not Figure 2/F. It should be Figure 2/C and 2/D.

LINE 158, there is not 2/G.

7. LINE 189, CpG

8. Please spell out the acronym SOEC.

9. What equations were used to fit curves to obtain EC50? LINE 239 only describes the software model. The curves are not reach to plateau and therefore, the EC50 values may not be accurate.

10. LINE 330 (1,40) to [1, 4].

11. LINE 385 Please spell out CRAC channels.

12. LINE 552 4.56?

13. LINE 87, References 33 and 34 are nothing to do with Facskin.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

In this study, Bajnok et al. investigated various type of human peripheral B cell responses against different stimuli through calcium flux. Although the point of author’s observation seems interesting, I have identified a number of mismatches between the figures, figure legends, and figure citations in the main text. For instance, in Figure 2A, the legend states that it shows the prevalence (%) of surface -immunoglobulin expression in B cell subsets, but the main text describes it as calcium flux of naïve B cells by stimulating with various stimuli. Moreover, there is no explanation of heat map kinetics parameters in the text around line 112, despite the fact that Figure 6 was cited along with Figure 2 at that point. Additionally, the order of figures in the main text starts from figure 2 instead of 1, which made me confused. Figure number should be ordered based on their appearance in the manuscript. As a result of these mismatches, I found it difficult to comprehend the content of this manuscript.

Author Response

In this study, Bajnok et al. investigated various type of human peripheral B cell responses against different stimuli through calcium flux. Although the point of author's observation seems interesting, I have identified a number of mismatches between the figures, figure legends, and figure citations in the main text. For instance, in Figure 2A, the legend states that it shows the prevalence (%) of surface -immunoglobulin expression in B cell subsets, but the main text describes it as calcium flux of naïve B cells by stimulating with various stimuli. Moreover, there is no explanation of heat map kinetics parameters in the text around line 112, despite the fact that Figure 6 was cited along with Figure 2 at that point. Additionally, the order of figures in the main text starts from figure 2 instead of 1, which made me confused. Figure number should be ordered based on their appearance in the manuscript. As a result of these mismatches, I found it difficult to comprehend the content of this manuscript.

Thank you for your suggestions; we have made the following corrections based on your recommendations. Figure 1 was previously cited in the 'Methods' section, and as this appears later in this journal format, the correct order of Figures was lost. Figure 1 is now cited at the end of the 'Introduction', where the experiments are described. Regarding Figure 2, a previous version was incorrectly inserted into the manuscript. Please see the updated and corrected Figure 2 and the corresponding legend. The Heat map, Figure 6, has been renamed Figure 3. Consequently, Figures 3, 4, and 5 are now Figures 4, 5, and 6, respectively. Figure 3 has been modified to match the flow of the text. The text of the 'Results’ section has been reorganized to correspond to the order of the Figure everywhere.

Round 2

Reviewer 1 Report

Good day,

  thanks to the authors for the changes they made. However, I must insist on the scholarly way of writing the publication

Introduction

Material and methods

The results

Discussion

Conclusion

Certainly your reading is engaging, but it does not allow to repeat the experiments you present. Only the collection of material and its preparation is not sufficiently described. Just as the methodology is not described - tools used, diagnostics and chemicals used.

Please supplement the work with these items.

Author Response

Thank you for your response. Upon consulting the handling Editor , this was their response:  

"Thanks for your mail. According to our journal’s rule, the correct order
of sections is
1. Introductions
2. Results
3. Discussion
4. Materials and Methods
5. Conclusions

Please adjust the section order accordingly, including reference, Table,
and Figure numbers."

The order of sections we used is according to the official template of IJMS and not up to the authors to decide, therefore, we were not able to change it. 

To the other question, we uploaded a detailed protocol in the supplementary material during the previous submission and now referenced it in the Materials and Methods section following the Discussion. I will now also send it as an attachment here. Could you please revise this protocol and let us know if there is any additional information we should add to it?

Thank you for your work, constructive criticism and thorough inspection of this manuscript, the quality of the manuscript was improved by your suggestions.

Kind regards,

Author Response File: Author Response.docx

Reviewer 3 Report

The authors revised the manuscript and corrected what I pointed out. This manuscript is well written. Results were carefully described and well discussed. The protocol was also described in detail. The contents are very interesting and provide a novel methodology to dissect a range of B cell biology. This study will contribute to understanding physiology and pathology of B cells. I’m satisfied with this manuscript.

Author Response

Thank you for your review. We feel that the reviewers' comments really added to the quality of the manuscript and are very grateful for their thorough work.

Round 3

Reviewer 1 Report

Dear authors,

thank you for incorporating the previous comments

and

please make the following corrections

p. 5 addition of description of units for measured quantities in all graphs. If "relative parameter value" is used, fill it in as it was calculated.

Furthermore, I would have one question about the basic concept - do you think that B cells cannot be affected by interaction with other cells, for example platelets?

Author Response

Dear Reviewer,

Thank you for your thorough revision and helpful comments.

Addition of description of units for measured quantities in all graphs. If "relative parameter value" is used, fill it in as it was calculated.

Thank you for this suggestion, to avoid confusion, the Y axis legend was changed from relative parameter value to Standardized Fluo-4 MFI on all Figures and the following description was added to the Figure legends:

“Standardized Fluo-4 MFI: Standardized Fluo-4-AM median fluorescence intensity, where the baseline absolute MFI value of Fluo-4 AM is standardized to 1 and relative parameter alterations are shown on the Y axis. “

Furthermore, I would have one question about the basic concept - do you think that B cells cannot be affected by interaction with other cells, for example platelets?

This is an interesting question. One benefit of this method is actually creating a more “natural” environment for immune cell activation as PBMCs compared to sorted samples for example. However, unspecific binding and activation must of course be avoided, and we screened for this during several steps. Most importantly, we recorded control curves without the addition of any activating agent to check the stability of the Fluo-4 signal and screen for any unspecific activation due to the experimental setup (for example due to mechanical stimuli or degradation of the dye). We found no alterations in these curves at all, Fluo-4 signals remained stable throughout the entire experiment. To address the question of thrombocytes, we found very few thrombocytes in our samples. This could be due to the isolation method (using Ficoll-Paque), where it has been shown that the double washing step following the isolation removes the overwhelming majority of thrombocytes. We checked all samples under the microscope for viability as well and found that PBMCs were not covered with thrombocytes.  Furthermore, we found that only very specific stimuli – via the BCR – could generate sufficient calcium signal for recording. We couldn’t even activate B cells using mitogens (PWM) or other receptors such as IL-4 or CD40. Therefore, we believe that unspecific binding derived activation in this experimental setup is negligible.