-->PONE-D-26-16996-->-->An optimized three-laser 27-color spectral flow cytometry panel for multi-organ profiling in mice-->-->PLOS One

Dear Dr. Lim,-->-->

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Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Partly

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: No

Reviewer #2: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript 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

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-->4. Is the manuscript 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 note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. 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 a valuable high‑dimensional flow cytometry panel, but few scientific issues require attention before publication – most notably the contradictory competing interest statement. The authors should address the unconventional ILC gating and provide additional validation for the neuronal marker CD56 in mouse tissues.Grammatical and formatting errors are mostly minor; they can be corrected during proofreading.

Reviewer #2: In the current manuscript the authors describe the development and optimization of a 27-color spectral flow cytometry panel using a 3-laser configuration for multi-organ immune profiling in mice. The study is technically relevant and addresses a practical gap in murine high-dimensional cytometry using widely accessible instrumentation. However, several concerns should be addressed prior to consideration for publication:

• The development of a 27-color panel on a 3-laser system is technically valuable. However, the manuscript would benefit from clear benchmarking against existing OMIPs and high-parameter spectral panels. The authors should better articulate the unique advantages and positioning of their panel.

• The fluorochrome assignment strategy (e.g., brightness index, spreading error) is described, but quantitative criteria guiding these decisions are insufficiently detailed. Providing explicit thresholds or a structured framework would improve reproducibility.

• The overall gating strategy is comprehensive and logically structured. However, the definition of mast cells and basophils requires clarification, as basophils are CD117- while mast cells are CD117+ (Fig. 1A). The current presentation may lead to misclassification.

• The identification of mScarlet+Ly6C+ monocytes across tissues is an interesting observation. However, this finding is not mechanistically explored and is currently overinterpreted as a biological conclusion rather than presented as an observational result. Additional validation or more cautious interpretation is recommended.

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Reviewer #1: Yes:  Natarajan Badri Narayan

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PLOSOne 27 color panel.docx

An optimized three-laser 27-color spectral flow cytometry panel for multi-organ profiling in mice

PONE-D-26-16996R1

Dear Dr. Jaechul Lim,

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.

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Kind regards,

Srinivasa Reddy Bonam

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: I Don't Know

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-->4. Have the authors made all data underlying the findings in their manuscript 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

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-->5. Is the manuscript 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 note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->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: This is a methodologically sound, well-executed, and genuinely useful technical resource paper, and I would recommend acceptance after minor revisions.

Core scientific contribution is real and addresses a genuine gap:

The central claim holds up: there is a documented scarcity of high-dimensional murine panels that work on standard 3-laser instruments, and the existing alternatives (OMIP-104, OMIP-105, and similar) either require 5-laser systems or are restricted to single tissues (spleen, tumor). A 27-color panel that runs on the much more widely available 3-laser Aurora and spans eight tissues simultaneously is a meaningful and practically valuable advance for the field. This isn't an incremental panel tweak — it solves an accessibility problem that many labs without 4–5 laser instruments genuinely face.

Panel design methodology is rigorous and well-documented:

The fluorochrome assignment logic (antigen density vs. brightness inversely paired, SSM-guided redistribution of high-spread combinations, iterative practical adjustment for reagent availability) follows established best practice (OMIP-069 framework) and is clearly described with enough granularity to be reproducible — exactly what a methods-focused OMIP-style paper should deliver. The titration strategy, FMO control use, and tissue-specific autofluorescence extraction are all appropriately justified and documented in the supplementary figures.

The authors responded to peer review in good faith and substantively

This is worth weighing heavily in a positive recommendation. Across both reviewers, the authors:

Added a full FDR-corrected statistical reanalysis rather than dismissing the multiplicity concern

Walked back overstated language ("glucocorticoid-producing" → "Cyp11b1-expressing"; "dynamic" → "acute") rather than defending indefensible claims

Disclosed the COI properly once flagged, rather than minimizing it

Added new validation data (Fig R1–R3) directly addressing specificity and cross-tissue gating concerns

Engaged with even minor terminology critiques (spectral unmixing vs. compensation, FcRI standardization) with full corrections

This is the behavior we want to see in a revision cycle, substantive engagement rather than perfunctory point-by-point deflection.

Appropriate epistemic humility in the Discussion

The limitations section is honest and complete: it explicitly flags the lack of intracellular/functional readouts, the female-only mouse cohort, the coarse-grained lineage definitions, and residual spectral spreading. This is exactly the kind of self-aware limitations section that strengthens rather than weakens a methods paper — it tells future users of the panel precisely where its boundaries are.

Biological validation (poly(I:C) model) is a credible proof-of-concept, not overreach once corrected

Using poly(I:C) as a systemic inflammatory challenge to demonstrate the panel can resolve coordinated multi-tissue immune remodeling is an appropriate and interpretable validation strategy. The finding of compartment-specific T cell dynamics (peripheral reduction vs. thymic stability) and the discovery of Ly6C⁺ monocyte Cyp11b1-reporter activity are interesting observations that, now properly hedged as reporter-level findings rather than confirmed steroidogenesis, are reasonable and additive to the literature.

Reviewer #2: (No Response)

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-->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: Yes:  Natarajan Badri Narayan

Reviewer #2: No

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