These are important protocols that will be useful to the field. Harmonisation of tissue isolation of DCs is important for cross-study comparisons.

The authors should note in the text that usually transcriptomics and functional assays should accompany flow cytometric phenotyping to accurately define DC subsets, particularly in disease/purturbed conditions.

IN general the work would benefit from some unification of introductory sections to reduce repetition and present more focused chapter-specific information.
The hierarchical numbering strategy needs to be made consistent throughout.
Check that terminology is consistent through the protocols (eg DC1 or cDC1, DC or DCs)
Acknowledgement sections need to be completed

More specific comments/suggestions:
Abstract
What does ‘generation’ (second line) mean in this context?
The title states ‘non-lymphoid tissue’ DCs but the abstract says ‘from lymphoid organs and various non-lymphoid tissues’ and section 6 regards DC from tumor-draining lymph nodes. Please be consistent

1.1 Lung tissue preparation
Check English and grammar throughout. Remove the use of ‘e.g.’ in the introduction
3.2 Preparation of lung DCs – specify this is for lung tissue (not BALF)
2. What is the maximum size of lung tissue sample for this protocol?
4. Define the approx. size of ‘very small pieces’
10 and 12. What temperature is the PBS added in these steps (eg RT, cold?)
12. Please define ‘proper’ in this step
13 (and in previous steps) writing ‘pre-warmed’ is a little confusing for a RT step. It would be clearer to just say room temperature (RT) and omit ‘pre-warmed’.
15. Please confirm centrifugation speed – 1800g seems very fast (usually 300-1000g for density centrifugation)? If correct, please explain why the high speed.
16. These cells are not PBMC (peripheral blood mononuclear cells) – they are lung mononuclear cells.
17. Temp of PBS in this step and all subsequent steps?
20. Why does this have to be in the dark?

Pitfalls – to maximise viability, as well as keeping samples on ice, it might be sensible to use cold reagents after the density centrifugation step? Cells should be at room temp prior to this step.

1.2 Lung DC Flow
Intro – It is equally as important that lung DCs are able to initiate or maintain tolerance as generate immunity to tissue and environmental antigens in lung.

I’m not sure of the relevance of this section?
This ability of antigen presentation is one of the key features of DCs, which are often simply described as “professional antigen presenting cells” however it is not unique to them[4]. The capacity of DCs (and others) to efficiently present antigens frequently is measured using mixed lymphocyte reaction assays, were antigen-experienced DCs are co-cultured with T cells whose proliferation rate and cytokine response then is measured.

“While cDC1 are capable of cross-presentation of antibodies” – cross presentation of antigens, not antibodies.

In the final paragraph of the introduction it is also noteworthy that to define DCs in tissues and in aberrant states, phenotypic analysis (with a few surface markers) often needs to be substantiated by both transcriptomic and functional analyses.

There would be less repetition if this section was merged with the previous section 1.1 (ie preparation and flow of lung DCs)?

Step 6. Final dilution by volume of 5%?
7. (or Table 4) please specify the volume of each antibody used (in your hands) per sample (or per 50ul staining volume as stated in step 10). As written, with 13 antibodies, this would be 3.85ul of each antibody?
Can Clec9A be used in place of CADM1 (to avoid requirement for secondary antibody staining and consequential loss of cells)?

Table 6 – Live/Dead (not Life/Dead)

2.1 Preparation of cells from skin
Intro
“DC can be grouped according to ontogeny or function, strong stimulatory capacity for naive T cell being the cardinal functional feature of DCs, irrespective of their progeny (2, 3)”. Do the authors mean ‘ontogeny’ rather than ‘progeny’?

DNAse I: what dose “(dissolved in A.dest)” mean?

Skin preparation
Step 2/3 – what is the maximum size piece(s) of skin that can be used here?

Enzymatic digestion
3. Reword “incubate the skin pieces at 37oC etc”
4. What temperature should the R10 be? Cold is centrifugation step is cold?
6. As the proportion of dead cells can’t be changed during counting (!) the wording may be better as “(typically not more than 20-30%)”
7. please write as a sentence and suggest defining cell number per 5cm x 3cm skin piece, as defined in step 4.

Pitfalls
Please specify the approximate size of ‘tiny pieces’. Are these the 5mm x 5mm pieces in 3.2.7?

Top tricks – does the ability to detect and quantify cell surface antigens change with longer enzymatic digestion times?

2.2 Flow cytometric analysis of skin cells
Introduction – another important function of skin DC is to maintain tolerance.
Typo – “it is still a matter of discussion IF they are skin-resident immune cells”
“xxx-color flow panel” needs to be completed.

3.3 antibody staining
Steps 4, 7, 10. Are there centrifugation steps here? If so, what speed for how long?
7. What volume do you wash in?

Table 4 is labelled as DC mouse panel – is this not about human DCs?

The dilutions of the antibodies are stated in both Table 2 (the second Table 2 as there are two Table 2s) and Table 4 but there are some discrepancies eg CD40 FITC – is the dilution 1:50 or 1:500?

Data analysis
Typo ‘healthy’

Do the authors use autofluorescence to identify macrophages?
As LCs are defined by CD1a-bright and Langerin-bright expression, these are commonly used to define LCs in gating strategies. What is the rationale of using CD1c and langerin in the final gating here?
Which DCs are CD16+ (B)?
What are the CD11c+HLA-DR- cells (C)?
CD141 is not the most reliable marker for cDC1 in tissue due to the fickle nature of CD141 expression. Is there a reason the authors do not use a more specific cDC1 marker (XCR1, Clec9A) to identify this subset (or gate as CD11c-low which would also distinguish from CD141-bright cDC2)?

Pitfalls
Does length of enzymatic digestion alter antigen expression?

3.1 Human Gingiva
Figure 2 – please define in words ‘very small pieces’ (although the picture is helpful) eg 5mm x 5mm

The surgical steps perhaps need not be included here?

3.3 Preparation
1. To attain sufficient numbers of DC for analysis, the minimal size of a sample is 3x2mm?

Figure legends appear twice in this version

3.2 Flow analysis
Are there any B cells in the tissue?
cDC1 are usually CD1c+ in tissue (skin, lung etc). How does the CD1c expression on cells in gingiva compare to eg pDC and/or to isotype? Are they truly negative?
What are the CD1cneg/lowCD141- cells in the final plot?

1. Human intestine preparation and flow cytometry
   Detailed description
   Have cDC1 in intestines been characterised with other markers such as XCR1, Clec9A?

2. Human tumours
   “but differentiates under inflammatory conditions along a separate lineage from cDC1 and cDC2 (Dutertre et al., 2020; Ginhoux et al., 2022).” This has also been shown in steady state (Cytlak et al 2020)

3.2. is written as a ‘results’ section rather than a protocol.
Eg “Biopsies of at least 1cm3 from surgical specimens were collected” could be ‘Biopsies of at least 1cm3 are required for processing’

Tweezers would usually be termed ‘forceps’
Step 8, 10 etc. Suggest stating centrifugation speeds as RCF (or xg) so they are translatable to other centrifuges.
Are steps 13-16 to wash the cells twice? Are both washes necessary here and why?

Flow gating
Intro – how do transcriptomics help to classify tumour DCs?

Taking an initial high CD11c gate may exclude true cDC1 which are usually CD11c low in tissues (and blood).
Have the authors looked for cDC1 in the CD11c low population?
This question is relevant for the tumour draining LN analysis as well.

Figures – please label axes of bivariate plots with antigen (and fluorochrome), and in larger font. (also relevant to Tumour draining LNs)

Is it possible to show the same gating strategy across the different tumours? (also relevant to Tumour draining LNs)

How does the flow cytometric phenotyping compare between digestion and physical dissociation methods? (also relevant to Tumour draining LNs)

1. Tumor Draining LNs
   3.2 processing – again use language specific to protocol writing rather than results?
   Does step 4 need to be on ice or not?
2. Typo - Scrape the cut surface. What is the reason to do this?
   Again – state centrifugation speeds as RCF ( or xg)

Figures
Intro – Type ‘Mamma carcinoma’ – is this mammary carcinoma?

Table 5 – can the authors include phenotypic markers of resident and non-resident DCs?