Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors This paper reports an effective method for demonstrating the maturation stages of T cells and NK cells using 14-colour flow cytometry. The practical methods of cell isolation and flow cytometry analysis are well considered and described. It will benefit researchers who want to analyse these cell populations using multiparametric flow cytometry. The unfortunate aspect is that all of the data are from LPS-treated mice. There is no data or description comparing the control animals that did not receive LPS. The cellular composition of T cells and NK cells in normal mice may be familiar to professionals. However, to make it more constructive for researchers from diverse backgrounds, it is better to present results from normal mice and compare them with LPS-treated mice. It will help demonstrate the effectiveness of this flow cytometry protocol and make it more attractive to researchers.

Author Response

Please find our detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled: “Multiparametric flow cytometry panel for characterization of mouse T cell differentiation and NK cell maturation following inflammatory challenge” suggest a panel for the phenotypic characterization for T and NK cell population. It is a well written manuscript that includes detailed description of protocols, but there are some major and some minor concerns to be addressed before reconsidering it for publication.

• Taking into consideration the variety of panels published referring to T cells and NK cells the authors should better highlight in their manuscript what is the added knowledge that their protocols brings to the field.
• In lines 217-219, the authors claim that their protocol can be applied apart from BD FACSSymphony A3 also to any other multiparametric flow cytometer. Have they applied it to other instruments? If not, please justify or omit this statement.
• Please add a paragraph explaining how the panel was build. Was it based on an existing protocol and then was expanded? What were the criteria of choosing the fluorochromes (eg brightness, antigen density etc).
• Lines 558-564: Please clarify if the dead or damaged cells are excluded from cell counting and cell number for each sample is based on counting only live cells. If not please state what is the point of assessing the number of dead/damaged cells.
• Please check the references used across the manuscript. There are references that are not in consistence with the text. For example, references 12 and 41 do not seem to include the relevant information written in lines 58-59 and 433-435, respectively. Moreover, information in lines 568-570 is included in reference 43 rather than in 42, while reference 43 does not contain any information regarding CyTOF.

Author Response

Please find our detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

SUMMARY

This manuscript presents a detailed laboratory protocol for multiparametric flow cytometric immunophenotyping of mouse lymph node T and NK cell populations. The scientific rationale rests on the premise that lymph nodes serve as critical sites of adaptive immune coordination, and that resolving the phenotypic heterogeneity of T and NK cell subsets at single-cell resolution requires validated, reproducible methods. The authors address the need for a standardized approach that integrates tissue dissociation, panel design, compensation strategy, and gating logic into a single reproducible workflow. The approach combines anatomically guided surgical dissection of eleven bilateral and visceral lymph node sites from BALB/cAnNCrl female mice with mechanical homogenization and enzymatic digestion using collagenase IV, hyaluronidase, and DNase I. The resulting single-cell suspensions are stained with a 14-color antibody panel resolved on a BD FACSymphony A3 instrument. The panel simultaneously identifies CD4+ and CD8+ T cell differentiation states (naïve, central memory, effector memory, and effector subsets defined by CD62L and CD44) and five sequential NK cell maturation stages (defined hierarchically by CD122, CD244, NKp46, CD49b, Ly-49C, CD11b, and KLRG1). Fluorescence minus one (FMO) controls, compensation bead-based single-stain controls, and a fixable viability dye are incorporated as standard practice.

 

As a methods article, this manuscript occupies a well-defined but incremental space in the literature. Multiparametric flow cytometric immunophenotyping of mouse lymphocytes is not a novel undertaking, and the individual components of this protocol, enzymatic digestion of lymphoid tissue, CD44/CD62L-based T cell differentiation staging, and NK maturation series based on CD122, NKp46, CD49b, and CD11b, are each well-established. The novelty, such as it is, lies in the integration of these components into a single 14-color panel and in the inclusion of Ly-49C and KLRG1 alongside classical maturation markers to define a five-stage NK maturation hierarchy, which extends somewhat beyond the conventional four-stage model. The LPS challenge model provides a biologically motivated validation context rather than a conceptual advance. For a methods-focused journal such as Methods and Protocols, this level of novelty is appropriate, provided the procedural detail, validation data, and documentation are of sufficient quality to enable independent reproduction, which is where the manuscript has notable gaps.

MAJOR COMMENTS

Comment 1

The manuscript lacks an unmanipulated control group (naïve or PBS-injected mice). Figure 5 presents population frequencies exclusively from LPS-treated animals at day 14 after the initial injection. The absence of a comparator group makes it impossible to determine whether the distributions shown reflect LPS-induced immune remodeling or simply the baseline immunophenotypic composition of BALB/cAnNCrl lymph nodes. This matters directly for the stated purpose of the protocol, which the authors describe (line 581–582) as enabling monitoring of "the dynamic immune repertoire induced by inflammatory stimulation" and serving "as a positive control for assessing maturation or trained immune responses." A protocol paper that proposes LPS challenge as a positive control for immune activation must demonstrate that the assay detects a difference relative to an unmanipulated state. Without this, the validation data in Figure 5 provide no evidence that the panel resolves LPS-dependent immune changes. The authors should either include data from a naïve or vehicle control group in Figure 5, or substantially revise the framing of this section to reflect that Figure 5 describes only the steady-state immunophenotypic output of the assay in LPS-treated animals.

Comment 2

The sample size of n=3 biological replicates (Figure 5 legend, line 662–663) is insufficient to support the procedural performance claims made in the manuscript. The authors state (line 532–533) that the protocol yields approximately 50 × 10^6 cells with at least 80% viability per mouse, and they describe a gating recommendation of at least 100 events per rare population (line 434–435). No data are presented for cell yield or viability across preparations, and the reported frequencies for the rarest NK maturation subsets (Stages 4 and 5 in Figure 5C) do not include documentation of whether the 100-event threshold was met. For a protocol paper, quantitative characterization of reproducibility including the distribution of cell yield, viability, and subset frequency across independent preparations is central to the utility of the article. Three biological replicates from a single experimental cohort do not provide this characterization. The authors should expand the validation dataset or, at minimum, report the actual event counts for the rarest populations across all three mice and explicitly address whether the recommended minimum was achieved.

Comment 3

The operational definition of Stage 1 NK cells (Table 5 and lines 590–591) is scientifically problematic and is stated without adequate justification. Stage 1 is defined as CD45+CD3−CD122+CD244−/+ NKp46−. The use of CD244−/+ as a defining criterion, combined with the absence of lineage exclusion markers (Lin−) that are standard in NK progenitor identification, means this gate captures a heterogeneous population that likely includes non-NK lineage cells with CD122 expression, notably precursor T cells and ILC precursors that also express CD122 and are CD3−. The Introduction itself acknowledges (lines 75–78) that CLPs are Lin−, and that CD127 expression within Lin−CD244+ progenitors marks early lymphoid specification, yet no lineage exclusion step is included in the Stage 1 gate. The panel does not include CD127, B220, or other lineage markers that would disambiguate NK progenitors from contaminating populations. The authors should explicitly acknowledge this limitation, revise the label "Pre-NK / NK progenitor–like cells" to reflect that this population is operationally defined and not purified, and refrain from implying direct developmental correspondence with published NK progenitor stages without the lineage exclusion data to support that correspondence.

Comment 4

The Homogenization Buffer formulation described in Section 2.3 (lines 171–173) contains a concentration inconsistency that would compromise reproducibility. The collagenase IV stock is listed in Table 1 materials as 80 mg/mL and is prepared to a final concentration of 80 mg/mL in the Homogenization Buffer, which would imply no dilution. The same logic applies to hyaluronidase at 32 mg/mL. These figures are implausible as working concentrations for enzymatic dissociation of lymphoid tissue and suggest that the final working concentrations have been confused with the stock concentrations. Furthermore, the protocol states that homogenized lymph nodes are transferred to "a 50 mL centrifuge tube containing 5 mL of collagenase and hyaluronidase mixture" (line 358–360), implying a second addition of enzyme beyond the Homogenization Buffer. The relationship between these two enzyme applications is not explained, and the total final enzyme concentrations in the digestion reaction are not calculable from the information provided. This inconsistency must be resolved with explicit statement of stock concentrations, working concentrations, and final concentrations in the digestion reaction.

Comment 5

The statistical reporting in Figure 5 is incomplete and the sample size is inadequate for the claims made. The figure legend (lines 662–663) states that data represent arithmetic mean ± SEM, n=3. For bar graphs displaying percentages of rare populations, where individual values may differ by factors of two to three, presenting only mean ± SEM without displaying individual data points obscures the biological variability and makes it impossible to assess the reliability of the reported means. This is particularly consequential for NK maturation stages 4 and 5, where expected cell frequencies are low and inter-animal variability is likely to be substantial. Standard practice for small-n flow cytometry datasets requires presentation of individual biological replicates alongside summary statistics. The figure should be revised to display individual data points overlaid on the bar graphs.

Comment 6

The numbering discrepancy in the ethical approval statement represents a potential integrity concern. In Section 2.1 (line 138), the permission number is cited as U34401-3/2022/17, whereas the Institutional Review Board Statement (line 680) cites U34401-3/2022/11. Both numbers cannot be correct for the same approved protocol. The authors must verify and correct the permit number, ensuring that a single accurate number is reported consistently throughout the manuscript.

MINOR COMMENTS

Comment 1

Table 1 lists "Brilliant Stain Buffer" in the Antibody Master Mix formulation (Section 2.3, line 183) but this reagent does not appear in the materials list in Section 2.2. The manufacturer, catalog number, and working volume should be added to Section 2.2 to ensure completeness.

Comment 2

The DNase I entry in the materials list (Section 2.2, lines 146–147) is formatted without a number, breaking the numbered list sequence between items 2 and 4. This should be corrected for clarity and consistency.

Comment 3

Line 113 uses the phrase "robust positive control" to describe LPS. The word "robust" should be replaced with a more specific descriptor, for example, "well-characterized" or "pharmacologically defined" to conform to precise scientific language.

Comment 4

The gating strategy schematic in Figure 3 is difficult to read at publication scale due to small font sizes in the binary state labels below each gating step. The figure should be redrawn with sufficiently large text to be legible in the final printed or digital format without magnification.

Comment 5

Lines 55–57 define the four T cell differentiation subsets based on CD62L and CD44 expression, including an "effector" subset defined as CD62L−CD44. The biological interpretation of CD44-negative effector T cells is contested; this phenotype is more commonly associated with recent thymic emigrants or non-antigen-experienced cells in some contexts. The Discussion (lines 584–587) appropriately notes that CD11b and KLRG1 require cautious interpretation, but the CD62L−CD44− "effector" designation deserves a similarly qualified statement in the text or in Table 5.

 

 

Author Response

Please find our detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I greatly appreciate a sincere response from the authors. I fully understand the purpose of this manuscript. Additional descriptions are clear and relevant, highlighting the importance of these flow cytometry panels.

Author Response

Please find detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately replied to all my comments and fully addressed my concerns regarding their manuscript.

Author Response

Please find detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Homogenization buffer concentration contradiction (unresolved in the revised manuscript)

The authors acknowledged the error in their response letter and described the correction accurately: working solutions of 80 mg/mL collagenase IV and 32 mg/mL hyaluronidase, diluted to final concentrations of 3.2 mg/mL and 0.32 mg/mL in the digestion buffer. That correction appears in the first part of the Homogenization Buffer preparation section (lines 203–206). However, the immediately following sentence (lines 207–209) states that the digestion mixture should be prepared by dissolving collagenase IV to a final concentration of 80 mg/mL and hyaluronidase to a final concentration of 32 mg/mL — the same implausible values that were present in the original submission and that the reviewer flagged as the primary reproducibility defect. The authors' response letter and the revised manuscript are therefore inconsistent with each other. A laboratory attempting to reproduce this protocol still cannot determine which concentration to use, because both sets of values appear sequentially in the same preparation step. This is the single most consequential error in the manuscript for independent reproducibility, and it remains uncorrected.

Residual contradictory framing following the reframing of Figure 5 (partially unresolved)

In response to the comment regarding the absence of an unmanipulated control group, the authors chose to reframe Figure 5 as an example of panel application rather than a formal demonstration of LPS-induced immune remodeling. That reframing is scientifically acceptable for a methods paper and is correctly reflected in several passages of the revised manuscript, including lines 648–652, which explicitly state that the data should not be interpreted as a formal assessment of LPS-induced immune remodeling. However, the revision was not applied consistently throughout the document. Lines 652–655 retain a sentence from the prior version stating that the experimental design enables monitoring of the dynamic immune repertoire induced by inflammatory stimulation and serves as a positive control for assessing maturation or trained immune responses. This sentence directly contradicts the reframing the authors committed to in their response. The two statements cannot coexist in the same paragraph: one disclaims any inference about LPS effects, and the immediately following sentence reasserts that the data demonstrate LPS-driven immune monitoring. This internal contradiction must be resolved by removing the retained fragment from the prior version.

Incomplete reconciliation of tracked changes throughout the manuscript

The revised manuscript contains multiple passages and formatting errors that indicate the tracked-changes reconciliation from the prior version was not completed before submission. These are not trivial typographical issues. In the Materials section (lines 155–180), items carry dual numbering throughout the list, with entries formatted as 3.4, 4.6, 5.7, and so on, which means the numbering system is incoherent and a user following the protocol cannot reliably identify which item is being referenced. Section headings carry inconsistent numbers in the same document, with Reagent Preparation appearing as both 2.3 and 2.4 and Equipment appearing as both 2.4 and 2.5. The Discussion contains a paragraph on PI-based viability assessment that is reproduced nearly verbatim in two consecutive locations (lines 604–614 and 615–622), with only minor variation between the two versions. The same section contains two near-identical sentences describing T cell stratification by CD62L and CD44 (lines 656–663 and 663–668), again with the second being a retained fragment of the prior version rather than a deliberate revision. In a protocol paper, where the primary scientific deliverable is procedural precision, these errors are not cosmetic. A reader attempting to follow the Materials list encounters a broken numbering system, a reader following the Discussion encounters repeated and partially contradictory guidance, and an editor reviewing the document cannot determine which version of any given passage represents the authors' final intended text.

Author Response

Please find detailed responses to the Reviewer's comments in the attached Word document.

Author Response File: Author Response.pdf