Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells

Piotr Pawel Janas; Caroline Chauché; Patrick Shearer; Georgia Perona-Wright; Henry J. McSorley; Jürgen Schwarze

doi:10.1371/journal.pone.0297585

Peer Review History

Original SubmissionAugust 22, 2023
18 Sep 2023 Decision Letter - Dominique Heymann, Editor PONE-D-23-26951Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cellsPLOS ONE Dear Dr. Schwarze, 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 summarized below raised during the review process. Please submit your revised manuscript by Nov 02 2023 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. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Reviewer #1: Partly ****** 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 ****** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this work, authors described and compared protocols to isolate and study murine airway epithelial cells (AECs). In particular, they investigated the effects of digestion time and temperature and they showed that a long and cold (20h 4°C) dispase II digestion of murine lungs have a considerable positive impact on AEC yield, viability, and ability to form colonies in vitro compared to an established 1h 37°C dispase II digestion step. Finally, they proposed a modified workflow for efficient AECs isolation that improves recovery and culture of airway epithelial cells. To contribute to the principles of replacement, reduction, and refinement (3R) in AEC research, isolated primary AECs are used in a range of in vitro experimental systems. The workflow presented in this manuscript may therefore interest a wide range of readers studying lung AECs and their role in the biology of lung. The manuscript is clear and well-written. Nevertheless, the authors should perform the minor modifications below to be suitable for publication. 1- The introduction could describe why you have chosen a magnetic-based isolation of cells with the depletion of CD45pos and CD31pos cells before the positive selection of EpCAM-expressing cells. 2- Please homogenize the writing of CD45-CD31-EpCAM+. Sometimes you used CD45-CD31-EpCAM+ and CD45-CD31EpCAM+. In Materials and Methods 3- In the “Murine lung harvest and digestion” part, line 94 : what does mean (S4)? 4- In the “Primary AECs in vitro culture” part, please verify the description of the medium between lines 144 and 147. See also supplement S4 protocol part. For example, line 144, “Media was prepared” should be replaced by ““Media were prepared” ; line 145, 1µM A3801 does not seem correct. Do you mean #03801 medium ? What is 1µM ? ; line 146, CHIR9902 should be CHIR 99021. 5- In “immunofluorescent microscopy” part, line 155 and line 157, “an-ti” should be replaced by “anti”. 6- In “RNA isolation and qPCR” part, line 193 and line 194, “ul” should be replaced by “µl”. In the legends of Figure 7- In Fig 3b, you mentioned “Representative immunofluorescent micrographs (cold digestion)” line 308, but you have also shown results for hot digestion, please modify. 8- What is the magnification used for the images ? 9- What is “Top scale bar, 2mm”, line 310? 10- In Fig 3c, could you specify if the images concern cold or hot digestion condition ? In supplement S4 protocol 11- “Militenyi” should be replaced by “Miltenyi”. 12- In “recipes”, “4C°” should be replaced by “4°C”. 13- Please check step 28. Is the cell pellet resuspended in 100µl of airway epithelial growth media (as indicated in supplement S4 protocol) or 0.5ml of MACS buffer (as indicated in Materials and Methods). 14- Please add the coating step description before step 30. ****** 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 ******** [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". 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Revision 1
31 Oct 2023 Author Response We would like to thank you and the reviewer for your constructive comments. We have now fully revised the paper as detailed point-by-point below. Editor 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 revised the manuscript and confirm that it is prepared according to PLOS ONE’s style requirements as found in the pdf files shared by the editor. We also revised the file naming requirements and confirm that they follow the PLOS ONE’s guidelines. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. The data present in the manuscript is now uploaded and available at Edinburgh DataShare public repository. The data can be accessed via https://doi.org/10.7488/ds/7506 3. We note you have not yet provided a protocols.io PDF version of your protocol and/or a protocols.io DOI. When you submit your revision, please provide a PDF version of your protocol as generated by protocols.io (the file will have the protocols.io logo in the upper right corner of the first page) as a Supporting Information file. The filename should be S1_file.pdf, and you should enter “S1 File” into the Description field. Any additional protocols should be numbered S2, S3, and so on. Please also follow the instructions for Supporting Information captions [https://journals.plos.org/plosone/s/supporting-information#loc-captions]. The title in the caption should read: “Step-by-step protocol, also available on protocols.io.” Please assign your protocol a protocols.io DOI, if you have not already done so, and include the following line in the Materials and Methods section of your manuscript: “The protocol described in this peer-reviewed article is published on protocols.io (https://dx.doi.org/10.17504/protocols.io.[...]) and is included for printing purposes as S1 File.” You should also supply the DOI in the Protocols.io DOI field of the submission form when you submit your revision. If you have not yet uploaded your protocol to protocols.io, you are invited to use the platform’s protocol entry service [https://www.protocols.io/we-enter-protocols] for doing so, at no charge. Through this service, the team at protocols.io will enter your protocol for you and format it in a way that takes advantage of the platform’s features. When submitting your protocol to the protocol entry service please include the customer code PLOS2022 in the Note field and indicate that your protocol is associated with a PLOS ONE Lab Protocol Submission. You should also include the title and manuscript number of your PLOS ONE submission. The protocol is now uploaded the protocol to protocols.io. It can be accessed via dx.doi.org/10.17504/protocols.io.rm7vzxo68gx1/v1 . We are also enclosing the protocols.io pdf version of the protocol as S1_file.pdf. The caption for the S1_file can now be found at the end of manuscript. 4. Please include captions for your S4 at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 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In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”B. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. All figures are now part of submission to data repository Edinburgh Data Share (https://doi.org/10.7488/ds/7506) , and as such all the figures are already under the CC BY 4.0 license. Please see https://datashare.ed.ac.uk/bitstream/handle/10283/8524/license_text?sequence=2&isAllowed=y for license details. 6. 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. The reference list was reviewed and we confirm that none of the included citations were retracted to the best of our knowledge. Reviewer #1 1. The introduction could describe why you have chosen a magnetic-based isolation of cells with the depletion of CD45pos and CD31pos cells before the positive selection of EpCAM-expressing cells. We now explain in the introduction (lines 80-83) why we are performing a two-step MACS sorting of EpCAM cells. 2. Please homogenize the writing of CD45-CD31-EpCAM+. Sometimes you used CD45-CD31-EpCAM+ and CD45-CD31EpCAM+. The writing of CD45-CD31-EpCAM+ was corrected and unified across the manuscript. 3. In the “Murine lung harvest and digestion” part, line 94 : what does mean (S4)? The S4 refers to supplemental information 4 – the protocol that is now submitted to protocols.io per PLOS ONE guidelines. As editor requested for a sentence in materials and methods referring to protocols.io protocol, the brackets are now removed. 4. In the “Primary AECs in vitro culture” part, please verify the description of the medium between lines 144 and 147. See also supplement S4 protocol part. For example, line 144, “Media was prepared” should be replaced by ““Media were prepared” ; line 145, 1µM A3801 does not seem correct. Do you mean #03801 medium ? What is 1µM ? ; line 146, CHIR9902 should be CHIR 99021. We apologies for the typos. These were now corrected (updated lines 147-150). 5. In “immunofluorescent microscopy” part, line 155 and line 157, “an-ti” should be replaced by “anti”. Thank you for pointing these out. These are now corrected. Corrected updated lines 162 and 164. 6. In “RNA isolation and qPCR” part, line 193 and line 194, “ul” should be replaced by “µl”. 7. Thank you for pointing these out. These are now corrected. Corrected updated lines 197 and 198. 8. In Fig 3b, you mentioned “Representative immunofluorescent micrographs (cold digestion)” line 308, but you have also shown results for hot digestion, please modify. Thank you for pointing that out. The figure description now indicates both hot and cold digestion. Corrected updated lines 311. 9. What is the magnification used for the images ? The 20x magnification is now indicated in updated lines 317-318. 10. What is “Top scale bar, 2mm”, line 310? Thank you for pointing that out. The “Top scale bar, 2mm” is now removed. The figure description was corrected and indicates a single scale bar of 0.1mm. Updated lines 316. 11. In Fig 3c, could you specify if the images concern cold or hot digestion condition? The Fig 3c description now specifies that it concerns cold digestion condition. Updated line 320. 12. “Militenyi” should be replaced by “Miltenyi”. Thank you for pointing that out. The typo was corrected throughout the manuscript and S4 supplemental protocol. 13. In “recipes”, “4C°” should be replaced by “4°C”. The typo was corrected. Thank you for pointing it out. 14. Please check step 28. Is the cell pellet resuspended in 100µl of airway epithelial growth media (as indicated in supplement S4 protocol) or 0.5ml of MACS buffer (as indicated in Materials and Methods). Thank you for pointing out the discrepancy. Both the S4 protocol and the manuscript were corrected to indicate 0.5ml of supplemented airway epithelial growth media. Updated line 136. 15. Please add the coating step description before step 30. Following step is now present in the protocol: “30. Coat the well plate with well-plate coating solution and incubate for at least 4-8h at 37°C before use.” We warmly thank the Reviewer for their positive appraisal, which together with their constructive comments and suggestions have greatly helped us in submitting an improved manuscript to your Journal. We hope this will be received positively and look forward to your decision in due course. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0297585.r002
14 Dec 2023 Decision Letter - Dominique Heymann, Editor PONE-D-23-26951R1Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cellsPLOS ONE Dear Dr. Schwarze, 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. Please submit your revised manuscript by Jan 28 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. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Dominique Heymann, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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 ******** 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 ****** 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 ****** 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: N/A ****** 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 ****** 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: There are still some discrepancies in your protocols. Please modify. 1- Chemical product names are still not correct. Please check thoroughly all your files (manuscript, supplementary…). CHIR99021 from stemcell Tech should not be CHIR9902 or CHIR9901 (reviewed manuscript). A 83-01 from stemcell Tech should not be A8301 in S3 Fig (manuscript, line 391). 2- Do you really have 1% Pen/Strep in your MACS buffer (S4 protocol) ? It is not indicated in “Murine lung harvest and digestion” part (manuscript, line 118). In addition, in “Recipes” of S4 protocol, you should name this buffer “MACS buffer” instead of “MACS buffer WASH”. It is confusing. 3- In “Recipes” of S4 protocol, you did correct as requested the “4C⁰” in “Airway epithelial growth media“ part, but you have forgotten to correct the “4C⁰” in “Well-plate coating solution “ part. Please modify. 4- Thanks for adding as requested the coating step (step 30) in S4 protocol. However, it would have been better if you had not made a typo. “coate” should be replaced by “coat”. ****** 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 ******** [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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0297585.r003
Revision 2
5 Jan 2024 Author Response To Dr Dominique Heymann 04.01.2024 Dear Dr Heymann, Manuscript submission PONE-D-23-26951 “Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells” We would like to thank you and the reviewer for your constructive comments. We have now fully revised the paper as detailed point-by-point below. Our comments are in blue. Editor 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 revised the manuscript and confirm that it is prepared according to PLOS ONE’s style requirements as found in the pdf files shared by the editor. We also revised the file naming requirements and confirm that they follow the PLOS ONE’s guidelines. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. The data present in the manuscript is now uploaded and available at Edinburgh DataShare public repository. The data can be accessed via https://doi.org/10.7488/ds/7506 3. We note you have not yet provided a protocols.io PDF version of your protocol and/or a protocols.io DOI. When you submit your revision, please provide a PDF version of your protocol as generated by protocols.io (the file will have the protocols.io logo in the upper right corner of the first page) as a Supporting Information file. 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If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. All figures are now part of submission to data repository Edinburgh Data Share (https://doi.org/10.7488/ds/7506) , and as such all the figures are already under the CC BY 4.0 license. Please see https://datashare.ed.ac.uk/bitstream/handle/10283/8524/license_text?sequence=2&isAllowed=y for license details. 6. 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. The reference list was reviewed, and we confirm that as of 14/12/2023 none of the included citations were retracted based on information available on PubMed. Several references (6, 11, 32) were corrected so that they indicate an exact article, rather than a collection of articles. One reference (27) was added as a consequence of edits suggested by reviewer #1. 7. 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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Thank you for your suggestion to use PACE. We confirm that all figures were uploaded to PACE to make sure that they meet PLOS requirement. Reviewer #1 1. The introduction could describe why you have chosen a magnetic-based isolation of cells with the depletion of CD45pos and CD31pos cells before the positive selection of EpCAM-expressing cells. We now explain in the introduction (lines 80-83) why we are performing a two-step MACS sorting of EpCAM cells. 2. Please homogenize the writing of CD45-CD31-EpCAM+. Sometimes you used CD45-CD31-EpCAM+ and CD45-CD31EpCAM+. The writing of CD45-CD31-EpCAM+ was corrected and unified across the manuscript. 3. In the “Murine lung harvest and digestion” part, line 94 : what does mean (S4)? The S4 refers to supplemental information 4 – the protocol that is now submitted to protocols.io per PLOS ONE guidelines. As editor requested for a sentence in materials and methods referring to protocols.io protocol, the brackets are now removed. 4. In the “Primary AECs in vitro culture” part, please verify the description of the medium between lines 144 and 147. See also supplement S4 protocol part. For example, line 144, “Media was prepared” should be replaced by ““Media were prepared” ; line 145, 1µM A3801 does not seem correct. Do you mean #03801 medium ? What is 1µM ? ; line 146, CHIR9902 should be CHIR 99021. We apologies for the typos. These were now corrected (updated lines 147-150). 5. In “immunofluorescent microscopy” part, line 155 and line 157, “an-ti” should be replaced by “anti”. Thank you for pointing these out. These are now corrected. Corrected updated lines 162 and 164. 6. In “RNA isolation and qPCR” part, line 193 and line 194, “ul” should be replaced by “µl”. 7. Thank you for pointing these out. These are now corrected. Corrected updated lines 197 and 198. 8. In Fig 3b, you mentioned “Representative immunofluorescent micrographs (cold digestion)” line 308, but you have also shown results for hot digestion, please modify. Thank you for pointing that out. The figure description now indicates both hot and cold digestion. Corrected updated lines 311. 9. What is the magnification used for the images ? The 20x magnification is now indicated in updated lines 317-318. 10. What is “Top scale bar, 2mm”, line 310? Thank you for pointing that out. The “Top scale bar, 2mm” is now removed. The figure description was corrected and indicates a single scale bar of 0.1mm. Updated lines 316. 11. In Fig 3c, could you specify if the images concern cold or hot digestion condition? The Fig 3c description now specifies that it concerns cold digestion condition. Updated line 320. 12. “Militenyi” should be replaced by “Miltenyi”. Thank you for pointing that out. The typo was corrected throughout the manuscript and S4 supplemental protocol. 13. In “recipes”, “4C°” should be replaced by “4°C”. The typo was corrected. Thank you for pointing it out. 14. Please check step 28. Is the cell pellet resuspended in 100µl of airway epithelial growth media (as indicated in supplement S4 protocol) or 0.5ml of MACS buffer (as indicated in Materials and Methods). Thank you for pointing out the discrepancy. Both the S4 protocol and the manuscript were corrected to indicate 0.5ml of supplemented airway epithelial growth media. Updated line 136. 15. Please add the coating step description before step 30. Following step is now present in the protocol: “30. Coat the well plate with well-plate coating solution and incubate for at least 4-8h at 37°C before use.” 16. Chemical product names are still not correct. Please check thoroughly all your files (manuscript, supplementary…). CHIR99021 from stemcell Tech should not be CHIR9902 or CHIR9901 (reviewed manuscript). A 83-01 from stemcell Tech should not be A8301 in S3 Fig (manuscript, line 391). We apologies for these omissions. We have now corrected all indicated product name typos. We also reviewed both the manuscript and the supplementary file with following changes applied: • Manuscript line 150 changed DMH-1 to DMH1 • Manuscript line 152 changed CHIR9901 to CHIR99021 • Manuscript line 391 changed CHIR9902 to CHIR99021 • Manuscript line 391 changed A8301 to A 83-01 • Manuscript line 151 changed “Y27632” to “Y-27632” • Manuscript line 391 changed “Y27632” to “Y-27632” • S4 protocol – DMH-1 changed to DMH1 in material list and in recipes • S4 protocol – changed CHIR9901 to CHIR99021 in materials list • S4 protocol – changed CHIR9901 to CHIR99021 in recipes • S4 protocol – changed “Y27632 ROCK” to “Y-27632” in recipes 17. Do you really have 1% Pen/Strep in your MACS buffer (S4 protocol) ? It is not indicated in “Murine lung harvest and digestion” part (manuscript, line 118). In addition, in “Recipes” of S4 protocol, you should name this buffer “MACS buffer” instead of “MACS buffer wash”. It is confusing. We apologies for omitting Pen/Strep in the manuscript MACS buffer recipe. This is now corrected in the manuscript line 118 where we added “1% v/v Penicillin/Streptomycin (10,000 U/ml, Gibco)”. We apologise for confusing the reader by using “MACS buffer” and “MACS buffer wash” interchangeably. Naming was corrected and “MACS buffer WASH” in S4 supplement was changed to “MACS buffer”. 18. In “Recipes” of S4 protocol, you did correct as requested the “4C⁰” in “Airway epithelial growth media“ part, but you have forgotten to correct the “4C⁰” in “Well-plate coating solution “ part. Please modify. We apologise for the incorrect “4C⁰” in “Well-plate coating solution“ S4 supplement. This has been now corrected to “4C⁰”. 19. Thanks for adding as requested the coating step (step 30) in S4 protocol. However, it would have been better if you had not made a typo. “coate” should be replaced by “coat”. We apologise for the typo. “coate” is now corrected to “coat”. We warmly thank the Reviewer for their positive appraisal, which together with their constructive comments and suggestions have greatly helped us in submitting an improved manuscript to your Journal. We hope this will be received positively and look forward to your decision in due course. Yours Faithfully, Professor Jürgen Schwarze Corresponding Author Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0297585.r004
9 Jan 2024 Decision Letter - Dominique Heymann, Editor Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells PONE-D-23-26951R2 Dear Dr. Schwarze, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Dominique Heymann, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0297585.r005
Formally Accepted
17 Jan 2024 Acceptance Letter - Dominique Heymann, Editor PONE-D-23-26951R2 PLOS ONE Dear Dr. Schwarze, 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 Pr. Dominique Heymann Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0297585.r006

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