An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescence

Camilla S. A. Davan-Wetton; Trinidad Montero-Melendez

doi:10.1371/journal.pone.0306275

Peer Review History

Original SubmissionMay 14, 2024
4 Jun 2024 Decision Letter - Luca Pesce, Editor PONE-D-24-18320An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescencePLOS ONE Dear Dr. Montero-Melendez, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== I have sent you the manuscript for minor revisions since enough feedback has been gathered. These revisions focus on enhancing the references and the statistical analysis of the work. ============================== Please submit your revised manuscript by Jul 18 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript presents an alternative and optimized protocol for senescence detection through lipofuscin pigments visualization in cultured cells using the well-known marker Sudan Black B. Furthermore, the authors demonstrated that this optimized method enables the simultaneous detection of lipofuscin (with Sudan Black B) and other proteins using a standard immunofluorescence technique, offering an advantage over traditional beta-galactosidase-based methods. Overall, the manuscript is well-written, the authors provide sufficient details to replicate the protocol and the conclusions align with the experimental results presented. Some minor revisions are needed: 1. Although Sudan Black B is a well-established marker for lipofuscin, the authors proposed an optimized detection method that was tested only in human dermal fibroblasts. It would be beneficial to test the Sudan Black B staining procedure on other types of cells that can undergo senescence-induction treatment. 2. The correlation analysis significantly strengthens the protocol's reliability. However, it is unclear from the main text and figure legends what types of treatments were used for the analysis (BMS-470539 at 1, 5, 10, or 20 µM?). 3. As stated by the authors, the work aims to determine the technical reliability of the protocol; therefore, more details are needed about the data analysis in Figures 5 and 6. Besides the number of replicates, the authors should report the number of cells analyzed for each condition and experiment. Was the integrated density measured for each cell (after creating a binary mask) or for the whole image? Reviewer #2: The manuscript describes an optimized methodology to stain lipofuscin as a marker of cellular senescence via Sudan black-B, which can be detected either through brightfield and fluorescence microscopy. The protocol that the authors describe represents an improvement with respect to the state-of-the-art since it is easier and allows for the simultaneous staining of additional markers through immunohistochemistry protocols. The results are clear and well-structured and therefore I recommend the work for publication. The following are questions that arose while reading the manuscript and may suggest additional information or clarifications that could strengthen the paper. 1. My first comment refers to the entire manuscript, where I think some terms are not properly used. The authors refer to different fluorescence detection windows (in terms of wavelength) as GFP-, RFP-, Cy3-… channels, although none of these fluorescent proteins or dyes are used in the experiments. Commercial microscopes may name the set of filters and parameters of excitation and emission as GFP, RFP, Cy3 channels for simplicity, but I believe it is wrong to maintain this nomenclature in the paper. What the authors are referring to is indeed the detection window in which, for example, Cy3 can be detected and whose excitation and emission filters have been found to be able to detect a fluorescent signal also from Sudan black-B. I suggest to the authors to change the nomenclature or, otherwise, to state they will refer to the specific detection window as “GFP” or “RFP” channel for simplicity. 2. In the introduction, I suggest to add a few more references (e.g. line 47, 53, 59..). 3. In line 90-91, the word “stain” seems redundant. 4. Line 121: I would specify that the chemicals described are used to induce senescence. Moreover, since this manuscript proposes a methodology, I would give more details (e.g. is PBS titrated to a specific pH? Is 4% PFA diluted in PBS or distilled water?) together with providing the product codes. 5. Figure 4 is completely black, I guess the image should be similar to what is shown in Supp Fig S3. Something has maybe happened while uploading the figure? 6. Line 283-293: I don’t have clear if the correlation analysis has been performed on the same cells stained with SA-b-Gal and then with SBB, or if it has been done on two different plates. 7. Line 328: typo error: “this autofluorescence is quenched when lipofuscin IS stained with SBB” 8. Line 329: the term “replaced” is not correct. What you are describing is that SBB quenches the autofluorescence signal of the cells that can be normally visualized by setting the exc and em parameters as you do to detect GFP and RFP (that you refer to as GFP and RFP channels). In addition to this, you found out that lipofuscin bound to SBB itself emits fluorescence in the “Cy3 channel”, but these are two different effects of using SBB and the second one is not a replacement of the first one. 9. Line 391-392: in Figure2, the top panel is lipofuscin accumulation with SBB and the middle is SA-b-Gal, but in the legend top and middle are inverted. 10. Line 469: I am confused, in the images there are not A and B but in the legend there are. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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Revision 1
10 Jun 2024 Author Response Manuscript: PONE-D-24-18320 Rebuttal Letter Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We have now revised and amended our manuscript according to the guidelines above. 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ The ORCID iD for the corresponding author has now been validated in Editorial Manager. 3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. We have now deleted the ethics statement that we initially included in a section after the figure legends. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. We have revised the reference list and to the best of our knowledge, no retracted papers have been included. References are provided in Vancouver style, as per guidelines. Reviewer #1: The manuscript presents an alternative and optimized protocol for senescence detection through lipofuscin pigments visualization in cultured cells using the well-known marker Sudan Black B. Furthermore, the authors demonstrated that this optimized method enables the simultaneous detection of lipofuscin (with Sudan Black B) and other proteins using a standard immunofluorescence technique, offering an advantage over traditional beta-galactosidase-based methods. Overall, the manuscript is well-written, the authors provide sufficient details to replicate the protocol and the conclusions align with the experimental results presented. Some minor revisions are needed: 1. Although Sudan Black B is a well-established marker for lipofuscin, the authors proposed an optimized detection method that was tested only in human dermal fibroblasts. It would be beneficial to test the Sudan Black B staining procedure on other types of cells that can undergo senescence-induction treatment. The suitability of SBB staining for the detection of lipofuscin as a marker of senescence in various cells and tissues has been extensively reported. The original paper (Georgakopoulou et al, 2013) describing lipofuscin accumulation as a general marker of senescence, already provided evidence of the suitability of SBB staining to detect lipofuscin in various cells, like mouse lung fibroblasts, two osteosarcoma cell lines, lung adenoma cells and prostate hyperplasia tissues. Evangelou et al (2017) report the use of SBB staining on human epithelial cells. Other examples include ovarian interstitial gland cells (Diaz-Hernandez,2022) or cardiomyocytes (Li et al, 2021). We have now included a new paragraph and these references in the introduction. The scope of our work is to provide a step-by-step protocol optimised for the detection of lipofuscin in human fibroblasts. Our protocol can certainly be used as a starting point by other scientists who work on other cell types and other forms of senescence and provide optimization of timing and other settings which will likely be very cell-type and senescence-type specific, to be included in one single protocol as the one we report here. 2. The correlation analysis significantly strengthens the protocol's reliability. However, it is unclear from the main text and figure legends what types of treatments were used for the analysis (BMS-470539 at 1, 5, 10, or 20 µM?). The correlation analysis provided in figure 6 correspond to the staining quantification reported in figure 5 (i.e. cells treated with 1, 5, 10 and 20 μM BMS-4700539, or vehicle). Only for the quantification of ‘bright field’ lipofuscin and ‘fluorescent’ lipofuscin, the same set of cells were used. However, as the colour visible under a bright field microscope for SA-βgalactosidase and SBB stain are very similar, separate sets of cells were used to allow precise visualization and quantification of each marker. We have added a note to the legend for Figure 6 to clarify this point: “[…] The data used for this analysis correspond to the results presented in Fig 5 in which cells were treated with vehicle or with 1, 5, 10, 20 μM BMS-470539). […].” 3. As stated by the authors, the work aims to determine the technical reliability of the protocol; therefore, more details are needed about the data analysis in Figures 5 and 6. Besides the number of replicates, the authors should report the number of cells analyzed for each condition and experiment. Was the integrated density measured for each cell (after creating a binary mask) or for the whole image? In Figures 5 and 6, for each technical replicate, a minimum of 20 cells were analysed for every staining protocol. For measuring the intensity of the fluorescent SBB signal (S4 fig), the integrated density was calculated for the whole image and then normalised by dividing this value by the number of cells in the image. This information has been added to the figure legends (5, 6 and S4) and the methods section also clarified. Reviewer #2: The manuscript describes an optimized methodology to stain lipofuscin as a marker of cellular senescence via Sudan black-B, which can be detected either through brightfield and fluorescence microscopy. The protocol that the authors describe represents an improvement with respect to the state-of-the-art since it is easier and allows for the simultaneous staining of additional markers through immunohistochemistry protocols. The results are clear and well-structured and therefore I recommend the work for publication. The following are questions that arose while reading the manuscript and may suggest additional information or clarifications that could strengthen the paper. 1. My first comment refers to the entire manuscript, where I think some terms are not properly used. The authors refer to different fluorescence detection windows (in terms of wavelength) as GFP-, RFP-, Cy3-… channels, although none of these fluorescent proteins or dyes are used in the experiments. Commercial microscopes may name the set of filters and parameters of excitation and emission as GFP, RFP, Cy3 channels for simplicity, but I believe it is wrong to maintain this nomenclature in the paper. What the authors are referring to is indeed the detection window in which, for example, Cy3 can be detected and whose excitation and emission filters have been found to be able to detect a fluorescent signal also from Sudan black-B. I suggest to the authors to change the nomenclature or, otherwise, to state they will refer to the specific detection window as “GFP” or “RFP” channel for simplicity. We understand the point raised and this is something we considered while writing the manuscript. As this is a methodological paper and the exact materials and equipment needs to be detailed, we decided that it was more correct to use the same terminology used by the manufacturer (ThermoFisher) to name their blocks (or Light Cubes, as they call them), so readers would easily find the detailed specifications if required. We did not choose to use “GFP” or “RFP” for simplicity, but because these are the names used by the manufacturer (perhaps the manufacturer used them for simplicity in the first place!). That is why we indeed provide the detection window of all the blocks that we use (e.g. GFP (green, EX470/22 - EM525/50)), so anyone would easily address the suitability of another manufacturer’s microscope blocks. Furthermore, for all the blocks, we also provide the “colour” name used for each one (e.g. green, far-red, etc), see methods: “Cells were visualised using the EVOS FL Imaging System (ThermoFisher) at different channels depending on the experiments: Cy5 (far red, EX628/40 - EM685/40) was used to detect SBB-stained lipofuscin; Texas Red (red, EX585/29 - EM628/32) was used to detect Alexa Fluor 594 conjugated antibodies; DAPI (blue, EX357/44 - EX447/60) was used to detect nuclei and the channels GFP (green, EX470/22 - EM525/50), RFP (orange EX531/40 - EM593/40) were used to detect…” 2. In the introduction, I suggest to add a few more references (e.g. line 47, 53, 59..). A number of additional references have now been included in the introduction. 3. In line 90-91, the word “stain” seems redundant. We have replaced “stain” with “detect”. 4. Line 121: I would specify that the chemicals described are used to induce senescence. Moreover, since this manuscript proposes a methodology, I would give more details (e.g. is PBS titrated to a specific pH? Is 4% PFA diluted in PBS or distilled water?) together with providing the product codes. Further details have been provided and the catalogue numbers added to all reagents included in the manuscript. 5. Figure 4 is completely black, I guess the image should be similar to what is shown in Supp Fig S3. Something has maybe happened while uploading the figure? Yes, this happened because the figures provided to reviewers in the merged pdf built upon submission are in low resolution to make file size more manageable for reviewers. We noticed this issue and added a note to the editorial office during our submission. 6. Line 283-293: I don’t have clear if the correlation analysis has been performed on the same cells stained with SA-b-Gal and then with SBB, or if it has been done on two different plates. This issue was also highlighted by Reviewer#1. Please see response to Question-2 in page 2. 7. Line 328: typo error: “this autofluorescence is quenched when lipofuscin IS stained with SBB” This typo has been corrected. 8. Line 329: the term “replaced” is not correct. What you are describing is that SBB quenches the autofluorescence signal of the cells that can be normally visualized by setting the exc and em parameters as you do to detect GFP and RFP (that you refer to as GFP and RFP channels). In addition to this, you found out that lipofuscin bound to SBB itself emits fluorescence in the “Cy3 channel”, but these are two different effects of using SBB and the second one is not a replacement of the first one. We have now removed the term replaced. This sentence now reads as follows: “However, whilst the GFP signal is lost, we demonstrate here that a pronounced emission of signal in the far-red channel that is indeed derived from the Sudan black B-stained lipofuscin emerges.” 9. Line 391-392: in Figure2, the top panel is lipofuscin accumulation with SBB and the middle is SA-b-Gal, but in the legend top and middle are inverted. This error has now been corrected. 10. Line 469: I am confused, in the images there are not A and B but in the legend there are. This error has been corrected, the mention of A and B was mistaken. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306275.r002
15 Jun 2024 Decision Letter - Luca Pesce, Editor An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescence PONE-D-24-18320R1 Dear Dr. Montero-Melendez, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have sufficiently answered my queries. I therefore have no further comments or revisions. Reviewer #2: All my previous comments have been addressed properly, therefore I recommend publication of this revised version of the manuscript. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0306275.r003
Formally Accepted
4 Jul 2024 Acceptance Letter - Luca Pesce, Editor PONE-D-24-18320R1 PLOS ONE Dear Dr. Montero-Melendez, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Luca Pesce Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306275.r004

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