Protocol for murine multi-tissue deep immunophenotyping using a 40-color full-spectrum flow cytometry panel

Summary

The innate and adaptive immune systems, though often studied separately, interact deeply and respond to stimuli simultaneously, with leukocytes displaying a range of pro- to anti-inflammatory phenotypes. This protocol details a procedure for characterizing murine innate and adaptive immune phenotypes using a 40-color full-spectral flow cytometry panel. We describe steps for organ collection, sample preparation, immunofluorescent staining, and acquisition to reproducibly and cost-effectively study tissue-resident leukocytes, their subpopulations, and inflammatory status in various organs.

Graphical abstract

Highlights

• •Steps for the isolation of murine leukocytes from pancreas, liver, and lungs
• •Instructions for immunofluorescent staining of 40 markers
• •Steps to identify tissular leukocytes, their subpopulations, and inflammatory status

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The innate and adaptive immune systems, though often studied separately, interact deeply and respond to stimuli simultaneously, with leukocytes displaying a range of pro- to anti-inflammatory phenotypes. This protocol details a procedure for characterizing murine innate and adaptive immune phenotypes using a 40-color full-spectral flow cytometry panel. We describe steps for organ collection, sample preparation, immunofluorescent staining, and acquisition to reproducibly and cost-effectively study tissue-resident leukocytes, their subpopulations, and inflammatory status in various organs.

Before you begin

The protocol below describes the specific steps to isolate leukocytes from pancreas, liver, and lung. Other tissues have also been successfully tested, for some, the digestion protocol needs to be adapted. The staining panel is designed to identify most murine leukocytes infiltrating the tissues, their subpopulations, and inflammatory phenotypes (Table 1). To design this panel, we used the Cytek Full Spectrum Viewer (Cytek Biosciences) (https://spectrum.cytekbio.com/) to select fluorochromes with distinct emission spectra. This selection was based on the similarity index calculated by the Spectrum Viewer, ensuring minimal spectral overlap. The fluorochromes were then ranked based on their staining index (https://cytekbio.com/pages/fluorochrome-guides). These staining indexes were calculated using a Cytek Aurora 5L, but since comparable data for the Sony ID7000 are not readily available, the Aurora 5L serves as a reliable proxy. Similarly, the antigens were ranked according to expression levels. When possible, fluorochromes with high stain indexes were assigned to antigens with low expression, while those with low stain indexes were paired with highly expressed antigens. Of note, some new fluorochromes are only available for common lineage antigens. Finally, we avoided using fluorochromes with a similarity index greater than 0.7 for antigens co-expressed on the same cells. The complexity index of the final panel is 46.63. Each antibody was then titrated and, if necessary, other antibody/fluorochrome combinations were tested.

Table 1.

List of the markers used in the panel, and their description and purpose

Marker (common name)	Description	Purpose
CD103	Integrin	Phenotyping of T cells, tissue residency marker and conventional dendritic cell (cDC1) marker
CD117 (c-kit)	Stem cell factor receptor	Lineage marker of mast cells, hematopoietic progenitor cells
CD11b	Integrin, cell adhesion and migration	Myeloid-lineage marker
CD11c	Integrin, cell adhesion, phagocytosis, cell migration	Lineage marker of conventional dendritic cells
CD163	Scavenger receptor	Phenotyping of monocytes and macrophages
CD170 (SiglecF)	glycan-binding protein	Lineage marker of eosinophils
CD185 (CXCR5)	Chemokine receptor	Phenotyping of T and B cells
CD19	B cell co-receptor, B cell development	Lineage marker of B cells
CD197 (CCR7)	Chemokine receptor, homing to and organization in the secondary lymphoid organs	Phenotyping of T cells, naïve
CD206	Mannose receptor, endocytosis and phagocytosis	Phenotyping of macrophages
CD25	Alpha chain of the IL-2 receptor, to form the high affinity receptor complex	Phenotyping of T cells, Treg
CD278 (ICOS)	Co-stimulatory molecule, cell-cell signaling	Phenotyping of T cells, activation
CD279 (PD1)	T cell inhibitory receptor, immune checkpoint	Phenotyping of T cells, activation and exhaustion
CD3	TCR co-receptor	Lineage marker for pan T cells, NKT cells
CD335 (NKp46)	NK cell receptor	Lineage marker of NK cells
CD357 (GITR)	Co-stimulatory receptor	Phenotyping of Treg, functional marker
CD4	TCR co-receptor	Lineage marker for CD4+ T cells
CD44	Adhesion receptor, cell migration and cell motility	Phenotyping of T cells, activation
CD45	Leukocyte common antigen	Pan-hematopoietic marker
CD45R (B220)	Protein tyrosine phosphatase	Lineage marker of B cells and plasmacytoid dendritic cells (pDCs)
CD49d	Integrin, cell adhesion	Phenotyping of T cell and neutrophils
CD5	Co-stimulatory molecule	Phenotyping of T cell and B cells
CD62L	L-selectin, tethering/rolling receptor to extravasate blood circulation	All circulating leukocytes, migration
CD69	Regulator of infiltrating lymphocytes	Phenotyping of T cells, tissue residency marker, activation
CD8	TCR co-receptor	Lineage marker for CD8+ T cells
CD9	Tetraspanin-family transmembrane protein	Phenotyping of B cells
F4/80	EGF-TM7 receptor	Lineage marker of macrophages
FcεRIα	High-affinity IgE receptor	Lineage marker of basophils
FoxP3	Transcription factor, master regulator of Treg differentiation	Treg marker
I-Ad	MHC class II	MHC class II
IgD	Immunoglobulin D	Phenotyping of B cells
IgM	Immunoglobulin M	Phenotyping of B cells
KLRG1	Co-inhibitory receptor	Phenotyping of T cells and NK cells, activation
Ly6C	Lymphocyte antigen 6 complex, locus C	Monocytes, macrophages and lymphocyte subsets
Ly6G	Lymphocyte antigen 6 complex locus G6D	Lineage marker of neutrophils
TCRβ	β chain of the T cell Receptor complex of αβ TCR	Lineage marker for αβ T cells
TCRγδ	δ chain of the T cell Receptor complex of γδ TCR	Lineage marker for γδ T cells
TIM-4	Recognition and efferocytosis of apoptotic cells	Phenotyping of macrophages
XCR1	Chemokine receptor	Phenotyping of dendritic cells, conventional dendritic cell (cDC1) marker

The panel can still be expanded by adding extra markers needed to answer specific experimental questions, while carefully selecting fluorochromes with low similarity to the current panel and considering co-expression on the cells of interest. For example, the BB660 fluorochrome (BD Biosciences) is a very bright dye that can be added to the panel with minimal impact. The RB744 fluorochrome (BD Biosciences) is also a very bright dye but has some spectral similarity with the RB780 fluorochrome (similarity index 0.75) used for CD9. It can be used to add a marker that is not co-expressed with CD9.

This protocol takes advantage of overnight staining, which increases resolution and is cost effective. It also uses spectral flow cytometry, which allows for higher parameter panels but is also effective in subtracting tissular autofluorescence.

Institutional permissions

NOD/ShiLtJ mice were purchased from Charles River (Italy), further inbred in the SPF facility of the KU Leuven (Leuven, Belgium), and maintained under semi-barrier conditions according to protocols approved by the KU Leuven Animal Care and Use Committee (000/(GS1/GS2)Breeding-Mathieu). Please check relevant institutional and national guidelines and regulations and acquire the needed permission before performing this protocol.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-mouse CD45 BUV395 (clone 30-F11); 1:5,000	Thermo Fisher Scientific	Cat# 363-0451-82, RRID: AB_2925264
Anti-mouse CD357 (GITR) BUV496 (clone DTA-1); 1:4,000	BD Biosciences	Cat# 741191, RRID: AB_2870756
Anti-mouse CD62L BUV563 (clone MEL-14); 1:1,000	BD Biosciences	Cat# 741230, RRID: AB_2870784
Anti-mouse CD170 (Siglec F) BUV615 (clone 1RNM44N); 1:300	Thermo Fisher Scientific	Cat# 366-1702-82, RRID: AB_2925422
Anti-mouse F4/80 BUV661 (clone T45-2342); 1:500	BD Biosciences	Cat# 750643, RRID: AB_2874771
Anti-mouse CD49d BUV737 (clone R1-2); 1:400	BD Biosciences	Cat# 741713, RRID: AB_2871087
Anti-mouse CD3 BUV805 (clone 17A2); 1:300	Thermo Fisher Scientific	Cat# 368-0032-82, RRID: AB_2896066
Anti-mouse CD3 BUV805 (clone 145-2C11); 1:300	BD Biosciences	Cat# 749276, RRID: AB_2873651
Anti-mouse IgD BV421 (clone 11-26c.2a); 1:1,000	BioLegend	Cat# 405725, RRID: AB_2562743
Anti-mouse CD11b VioBlue (clone REA592); 1:300	Miltenyi	Cat# 130-113-810, RRID: AB_2726327
Anti-mouse γδ T cell receptor BV480 (clone GL3); 1:300	BD Biosciences	Cat# 746343, RRID: AB_2743663
Anti-mouse IgM BV510 (clone RMM-1); 1:300	BioLegend	Cat# 406531, RRID: AB_2650758
Anti-mouse IgM BV510 (clone R6-60.2); 1:300	BD Biosciences	Cat# 747733, RRID: AB_2872206
Anti-mouse CD8a Spark Violet 538 (clone QA17A07); 1:1,000	BioLegend	Cat# 155020, RRID: AB_2890706
Anti-mouse Ly-6C BV570 (clone HK1.4); 1:500	BioLegend	Cat# 128030, RRID: AB_2562617
Anti-mouse/rat XCR1 BV605 (clone ZET); 1:200	BioLegend	Cat# 148222, RRID: AB_2927815
Anti-mouse CD69 BV650 (clone H1.2F3); 1:200	BioLegend	Cat# 104541, RRID: AB_2616934
Anti-mouse/human KLRG1 BV711 (clone MAFA); 1:3000	BioLegend	Cat# 138427, RRID: AB_2629721
Anti-mouse CD5 BV750 (clone 53-7.3); 1:300	BD Biosciences	Cat# 747114, RRID: AB_2871865
Anti-mouse CD11c BV785 (clone N418); 1:1,000	BioLegend	Cat# 117336, RRID: AB_2565268
Anti-mouse I-Ad AF488 (clone 39-10-8); 1:300	BioLegend	Cat# 115008, RRID: AB_493147
Anti-mouse TCR beta AF532 (clone H57-597); 1:1,000	Thermo Fisher Scientific	Cat# 58-5961-82, RRID: AB_2811915
Anti-mouse/human CD45R/B220 Spark Blue 574 (clone RA3-6B2); 1:500	BioLegend	Cat# 103290, RRID: AB_2904275
Anti-mouse FceR1a BB630 (clone MAR-1); 1:500	BD Biosciences	Cat# 624294, RRID: Custom order
Anti-mouse CD19 BB700 (clone 1D3); 1:300	BD Biosciences	Cat# 566411, RRID: AB_2744315
Anti-mouse CD25 PerCP-eFluor710 (clone PC61.5); 1:3,000	Thermo Fisher Scientific	Cat# 46-0251-82, RRID: AB_2734935
Anti-mouse CD9 RB780 (clone KMC8); 1:300	BD Biosciences	Cat# 755580, RRID:
Anti-mouse CD279 (PD-1) PerCP-Fire806 (clone 29F.1A12); 1:1,000	BioLegend	Cat# 135262, RRID: AB_2941420
Anti-mouse CD185 (CXCR5) PE (clone L138D7); 1:200	BioLegend	Cat# 145504, RRID: AB_2561968
Anti-mouse/human CD44 Spark YG 593 (clone IM7); 1:500	BioLegend	Cat# 103078, RRID: AB_2892266
Anti-mouse CD103 PE-CF594 (clone M290); 1:1,000	BD Biosciences	Cat# 565849, RRID: AB_2739377
Anti-mouse CD197 (CCR7) PE-Cy5 (clone 4B12); 1:1,000	BioLegend	Cat# 120114, RRID: AB_2072905
Anti-mouse CD206 (MMR) PE-Fire700 (clone C068C2); 1:500	BioLegend	Cat# 141741, RRID: AB_2922468
Anti-mouse Tim-4 PE-Cy7 (clone RMT4-54); 1:1,000	BioLegend	Cat# 130010, RRID: AB_2565719
Anti-mouse Ly-6G PE-Fire810 (clone 1A8); 1:2,000	BioLegend	Cat# 127673, RRID: AB_2910290
Anti-mouse FOXP3 APC (clone FJK-16s); 1:300	Thermo Fisher Scientific	Cat# 17-5773-82, RRID: AB_469457
Anti-mouse CD335 (NKp46) eFluor 660 (clone 29A1.4); 1:300	Thermo Fisher Scientific	Cat# 50-3351-82, RRID: AB_10598664
Anti-mouse CD4 APC-Cy5.5 (clone GK1.5); 1:2,000	Abnova	Cat# MAB5925, RRID: AB_10558755
Anti-mouse CD117 (c-kit) R718 (clone ACK2); 1:3,000	BD Biosciences	Cat# 567841, RRID: AB_2916759
Anti-mouse CD278 (ICOS) APC-eFluor780 (clone C398.4A); 1:3,000	Thermo Fisher Scientific	Cat# 47-9949-82, RRID: AB_2744732
Anti-mouse CD163 APC-Fire810 (clone S15049I); 1:500	BioLegend	Cat# 155321, RRID: AB_2904294
Anti-mouse CD16/32 purified (clone 93); 1:100	BioLegend	Cat# 101302, RRID: AB_312801
Chemicals, peptides, and recombinant proteins
Bovine serum albumin (BSA)	Sigma-Aldrich	Cat# A7030-10G
RPMI 1640 medium, GlutaMAX supplement, HEPES	Gibco	Cat# 72400021
DPBS, no calcium, no magnesium	Gibco	Cat# 14190144
DPBS (10X), no calcium, no magnesium	Gibco	Cat# 14200067
MEM non-essential amino acids solution (100X)	Gibco	Cat# 11140035
Sodium pyruvate (100 mM)	Gibco	Cat# 11360039
Penicillin-streptomycin (10,000 U/mL)	Gibco	Cat# 15140122
Fetal bovine serum (FBS)	Gibco	Cat# 10270106
Percoll centrifugation media	Cytiva	Cat# 17089101
LIVE/DEAD fixable blue dead cell stain kit, for UV excitation	Thermo Fisher Scientific	Cat# L34962
BD Horizon Brilliant Stain Buffer Plus (BSB+)	BD Biosciences	Cat# 566385
UltraComp eBeads compensation beads (CompBeads)	Thermo Fisher Scientific	Cat# 01-2222-42
eBioscience Foxp3/transcription factor staining buffer set	Thermo Fisher Scientific	Cat# 00-5523-00
Collagenase D	Sigma	Cat# 11088866001
DNase I	AppliChem	Cat# A3778.0050
CaCl2	Merck	Cat# 105833
MgCl2	Merck	Cat# 102382
EDTA (0.5 M), pH 8.0, RNase-free	Invitrogen	Cat# AM9260G
Experimental models: Organisms/strains
NOD/ShiLtJ (FELASA specific-pathogen-free facility)	In-house breeding (originated from Charles River Italy)	https://www.criver.com/products-services/find-model/jax-nod-mice?region=3616
Software and algorithms
FlowJo v10.8 software	BD Life Sciences	https://www.flowjo.com/
Prism v10.2.3 software	GraphPad Software	https://www.graphpad.com/
Other
Mesh woven filters, NITEX	Sefar	Cat# SEFA03-100/44
Microtiter plates, clear, V bottom	Thermo Scientific	Cat# 611V96
Centrifuge	Hettich	ROTINA 420 R
LUNA-FX7	Logos Biosystems	Cat# L70001
MaxQ 4000 benchtop orbital shakers	Thermo Scientific	Cat# SHKE4000-8CE
Sony ID7000	Sony Biotechnology	Cat# LE-ID7000C

Materials and equipment

DNase I stock

For 6.47 mg/mL DNase I stock, dissolve 50 mg DNase I in 7.73 mL double-distilled water (ddH₂O). Store at −20°C in aliquots for up to 12 months.

BSA stock

For 100 mg/mL BSA stock, dissolve 10 g BSA in 100 mL ddH₂O. Filter/sterilize using a low-protein-binding 0.22 μm filter. Store indefinitely at −20°C.

Digestion medium

Reagent	Final concentration	Amount
RPMI 1640 medium, GlutaMAX Supplement, HEPES	N/A	379 mL
Heat-inactivated FBS	20% (v/v)	100 mL
MEM NEAA	1% (v/v)	5 mL
Na-pyruvate (100 mM)	1 mM	5 mL
MgCl2 (1 M)	2 mM	1 mL
CaCl2 (100 mM)	2 mM	10 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

WASH buffer

Reagent	Final concentration	Amount
RPMI 1640 medium, GlutaMAX Supplement, HEPES	N/A	465 mL
Heat-inactivated FBS	2% (v/v)	10 mL
Streptomycin/Penicillin (P/S, 10,000 U/mL)	5% (v/v)	25 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

FACS buffer

Reagent	Final concentration	Amount
1X PBS	N/A	493 mL
BSA (100 mg/mL)	1 mg/mL	5 mL
EDTA (0.5 M stock)	2 mM	2 mL
Total	N/A	500 mL

Store at 4°C for up to 6 months.

40% Percoll

Reagent	Final concentration	Amount
Percoll	40% (v/v)	200 mL
10X PBS	N/A	45 mL
ddH2O	N/A	255 mL
Total	N/A	500 mL

Store at room temperature for up to 6 months.

Step-by-step method details

Sample collection

Timing: 10 min per mouse

This steps details how to perfuse a mouse to reduce contamination of circulating immune cells in the organs and collect the organs of interest.

• 1.Add 5 mL ice-cold WASH buffer per well of a 6-well plate for each organ and keep the plate on ice.
• 2.Euthanize the mouse accordingly to the local ethical committee requirement.
• 3.
  Prepare materials for perfusion.

  • a.
    Scissors and tissue forceps.
  • b.
    24G needle with a 10 mL syringe.
  • c.
    Tissue paper.
  • d.
    Ice-cold PBS.
• 4.
  Mouse dissection and perfusion:

  • a.
    Open the mouse with a V-shape cut at the abdomen.
  • b.
    Subtract the skin coat.
  • c.
    Open the thorax.
  • d.
    Make a cut in the right atrium, and perfuse the mouse with 10 mL ice-cold PBS in the left ventricle.
  • e.
    Soak all blood with tissue paper (Figure 1A).

CRITICAL: Ensure that the blood has been correctly washed out of all organs by controlling the color of the lung and liver (Figure 1B). Perfuse with more volume if necessary.

• 5.Collect the pancreas, liver and lung from each mouse and put in the corresponding well.

Figure 1.

Tissue perfusion

The color of the lung (arrows) and the liver (dashed contour) (A) before perfusion and (B) after perfusion.

Preparation of single cell suspension from pancreas, liver, and lung

Timing: 2 h 30 min

This step describes the digestion of the different organs and processing to obtain single cell suspensions free of tissue debris (Figure 2).

CRITICAL: Except indicated otherwise, always keep the cells on ice or at 4°C.

• 6.
  For each organ:

  • a.
    Add 1 mL ice-cold WASH buffer in glass Petri plate and add the organ.
  • b.
    Cut in very small pieces with surgical blades until all pieces are sufficiently thin.
  • c.
    Add 5 mL of WASH buffer to rinse the Petri plate and to make sure all pieces are in suspension.
  • d.
    Take up all the liquid and pieces of organ and transfer to a 15 mL tube.
  • e.
    Wash the plate again with another 5 mL of ice-cold WASH buffer and transfer to the tube.
• 7.Centrifuge at 400 × g for 5 min.
• 8.Prepare the digestion medium by adding 2 mg/mL of collagenase D and 40 μg/mL of DNase I to the digestion buffer pre-warmed in a 37°C water bath.
• 9.Remove the supernatant, add 5 mL of the digestion medium (Step 8) and mix well.
• 10.Incubate horizontally at 37°C with continuous shaking at 250 rpm for 30 min.
• 11.Flush the lung suspension using an 18 G needle to dissociate aggregates and incubate the lung for another 30 min as in Step 10. For pancreas and liver proceed directly to Step 12.

Note: The next steps are common for all the organs. You may start processing the pancreas and liver, while the lung is still digesting.

• 12.Place the tubes on ice.
• 13.Gently flush the suspension using an 18 G needle to dissociate aggregates (Figure 3A) until no more pieces are visible (Figure 3B).
• 14.Add 5 mL of ice-cold WASH buffer.
• 15.Put a Nitex tissue onto a 15 mL tube and pass the homogenized tissue through it.
• 16.Centrifuge at 400 × g for 5 min, 4°C.
• 17.Put 4 mL of 40% Percoll in a new 15 mL tube.
• 18.Remove the supernatant and resuspend the pellet with 1 mL of 40% Percoll.
• 19.Transfer the cells resuspended in 40% Percoll to the prepared tube (Step 17) by carefully layering the cells onto the 4 mL Percoll (Figures 4A and 4B).
• 20.Centrifuge at 400 × g for 10 min, at room temperature.
• 21.Carefully aspirate the supernatant without disturbing the pellet (Figures 5A and 5B).

CRITICAL: Do not pour off the Percoll.

• 22.Resuspend the pellet in 1 mL ice-cold FACS medium and count the cells accordingly to the lab SOP.
• 23.Add 9 mL ice-cold FACS medium.
• 24.Centrifuge at 400 × g for 5 min, at 4°C.
• 25.Carefully aspirate the supernatant.
• 26.Resuspend the pellet with 1 mL ice-cold FACS medium per 10⁷ cells.

Figure 2.

Schematic representation of the workflow for the preparation of single cell suspension

Figure 3.

Tissue dissociation

Pancreatic tissue after digestion (A) is physically dissociated using a 1 mL syringe and an 18G needle (B).

Figure 4.

The cell suspension is layered on top of the 40% Percoll

(A) Pancreas cell suspension.

(B) Liver cell suspension.

Figure 5.

Cell pellet after Percoll centrifugation

(A) Pancreas.

(B) Liver.

Cell surface staining

Timing: 1 h 30 min

In this step we will explain the fluorescent conjugated antibody staining including Fc receptor blocking, live/dead staining, and cell surface staining of 7 surface proteins that are only recognized or give better results in their native form.

CRITICAL: Always keep the cells on ice or at 4°C.

• 27.Add 200 μL of each cell suspension to a V-bottom (conical) 96-well plate (2 × 10⁶ cells).

Note: U-bottom 96-well plates can be used; however, in our experience, V-bottom 96-well plates make it easier to remove the supernatant without disturbing the pellet.

• 28.Centrifuge at 400 × g for 5 min, at 4°C.
• 29.
  Prepare the surface staining mix.

  • a.
    99 μL FACS buffer + 1 μL anti-mouse CD16/32 purified antibody (Fc receptor blocking) per sample.

    Note: Fc receptor, like FcγI, FcγII and FcγIII can be expressed by different leukocytes, with increased expression when activated. This may result in aspecific binding of some IgG isotypes, making interpretation of the results difficult.

  • b.
    Add the amount of each antibody per sample (Tables 2 and S1).
• 30.Just before use, spin down the surface staining mix at 10,000 × g for 5 min, at 4°C to remove antibody aggregates.¹
• 31.Discard the supernatant from the plate.

Note: The supernatant in the plate can be discarded using an aspirating manifold. However, we prefer to remove the supernatant by inverting, flicking, and blotting the plate on an absorbent surface, which reduces the risk of aspirating the pellet.

• 32.Resuspend cell pellets with 100 μL of surface staining mix.

CRITICAL: Leukocytes from tissue have a higher and specific autofluorescence. To extract this autofluorescence, we need a reference. For each organ, add one well with 200 μL of cell suspension (2 × 10⁶ cells) that will be processed the same way as the samples, but will not be stained. During the staining steps, incubate in the same medium, but without any antibody or dye. If you anticipate not having enough cells, use an extra mouse. It is important to treat the unstained samples in the same way as the stained samples, as this may affect the autofluorescence signature.

• 33.Mix well and incubate for 1 h in a refrigerator (2−8°C).

Note: 1-h incubation improves the resolution compared to the “standard” 30-min incubation.²

• 34.Add 100 μL of ice-cold FACS medium.
• 35.Centrifuge at 400 × g for 5 min, at 4°C.
• 36.Discard the supernatant.
• 37.Add 200 μL of ice-cold FACS medium.
• 38.Centrifuge at 400 × g for 5 min, at 4°C.
• 39.Discard the supernatant.

Table 2.

Surface staining mix

Antibody	Dilution factor	Volume per sample
LIVE/DEAD Fixable Blue	500	0.24 μL
BUV563-conjugated anti-mouse CD62L	1,000	0.12 μL
BV605-conjugated anti-mouse XCR1	200	0.60 μL
BV650-conjugate anti-mouse CD69	200	0.60 μL
AF532-conjugated anti-mouse TCRβ	500	0.24 μL
Spark Blue 574-conjugated anti-mouse B220	500	0.24 μL
BB700-conjugated anti-mouse CD19	300	0.40 μL
PE-conjugated anti- mouse CXCR5	200	0.60 μL

Cell fixation

Timing: 45 min

In this step, the cells will be fixed and permeabilized.

• 40.Resuspend the cells with 100 μL of eBioscience Foxp3 fix/perm buffer.

Note: You need to mix the Foxp3 fix/perm buffer concentrate with the diluent, accordingly to manufacturer’s indications in advance (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F00-5521.pdf).

• 41.Incubate for 30 min at room temperature in the dark with a lid.

CRITICAL: Longer fixation time leads to decrease staining of some epitopes.

• 42.
  Prepare the intracellular staining mix during the incubation time by mixing.

  • a.
    12 μL of eBioscience Foxp3 10X perm buffer + 12 μL BSB+ + 96 μL ddH₂O per sample.
  • b.
    Add the amount of each antibody per sample (Tables 3 and S1).
• 43.Add 100 μL of ice-cold 1x eBioscience permeabilization buffer to each well.

Note: eBioscience permeabilization buffer has to be reconstituted accordingly to manufacturer’s instructions in advance (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2F00-5521.pdf).

• 44.Centrifuge at 400 × g for 5 min, at 4°C.
• 45.Discard the supernatant.
• 46.Add 200 μL of ice-cold 1x eBioscience permeabilization buffer to each well.

Table 3.

Intracellular staining mix

Antibody	Dilution factor	Volume per sample
BUV395-conjugated anti-mouse CD45	5,000	0.024 μL
BUV496-conjugated anti-mouse GITR	4,000	0.030 μL
BUV615-conjugated anti-mouse SiglecF	300	0.400 μL
BUV661-conjugated anti-mouse F4/80	500	0.240 μL
BUV737-conjugated anti-mouse CD49d	400	0.300 μL
BUV805-conjugated anti-mouse CD3 (clone 17A2)	500	0.400 μL
BUV805-conjugated anti-mouse CD3 (clone 145-2C11)	500	0.400 μL
BV421-conjugated anti-mouse IgD	1,000	0.120 μL
VioBlue-conjugated anti-mouse CD11b	300	0.400 μL
BV480-conjugated anti-mouse TCRgd	300	0.400 μL
BV510-conjugated anti-mouse IgM (clone RMM-1)	300	0.400 μL
BV510-conjugated anti-mouse IgM (clone R6-60.2)	300	0.400 μL
Spark Violet 538-conjugated anti-mouse CD8	1,000	0.120 μL
BV570-conjugated anti-mouse Ly6C	500	0.240 μL
BV711-conjugated anti-mouse KLRG1	3,000	0.040 μL
BV750-conjugated anti-mouse CD5	300	0.400 μL
BV785-conjugated anti-mouse CD11c	1,000	0.120 μL
AF488-conjugated anti-mouse I-Ad	300	0.400 μL
AF532-conjugated anti-mouse TCRb	500	0.240 μL
Spark Blue 574-conjugated anti-mouse B220	500	0.240 μL
BB630-conjugated anti-mouse FcεRIα	500	0.240 μL
PerCP-eFluor710-conjugated anti-mouse CD25	3,000	0.040 μL
RB780-conjugated anti-mouse CD9	300	0.400 μL
PerCP-Fire806-conjugated anti-mouse PD1	1,000	0.120 μL
Spark YG 593-conjugated anti-mouse CD44	500	0.240 μL
PE-CF594-conjugated anti-mouse CD103	1,000	0.120 μL
PE-Cy5-conjugated anti-mouse CD197 (CCR7)	1,000	0.120 μL
PE-Fire700-conjugated anti-mouse CD206	500	0.240 μL
PE-Cy7-conjugated anti-mouse TIM-4	1,000	0.120 μL
PE-Fire810-conjugated anti-mouse Ly6G	2,000	0.060 μL
APC-conjugated anti-mouse FoxP3	300	0.400 μL
eFluor 660-conjugated anti-mouse NKp46	300	0.400 μL
APC-Cy5.5-conjugated anti-mouse CD4	2,000	0.060 μL
R718-conjugated anti-mouse CD117 (c-kit)	3,000	0.040 μL
APC-eFluor780-conjugated anti-mouse ICOS	3,000	0.040 μL
APC-Fire810-conjugated anti-mouse CD163	500	0.240 μL

Surface and intracellular overnight antibody staining

Timing: 18 h

In this step, we stain both fixed surface proteins and intracellular proteins. As the cells are fixed, we can prolong the incubation time, without risk of cells changing their status or dying. This allows for the use of very low amounts of antibodies (cost-effective), while improving detection sensitivity.²

• 47.Centrifuge the plate at 400 × g for 5 min, at 4°C.
• 48.Discard the supernatant.
• 49.Spin down the intracellular staining mix at 10,000 × g for 5 min, at 4°C to remove antibody aggregates.¹
• 50.Resuspend the cells with 100 μL of intracellular staining mix (Step 42).
• 51.Mix well and incubate overnight in a refrigerator (2−8°C).

Note: Staining resolution increase up to 16 h of incubation and stays stable at least until 24 h.²

• 52.
  Prepare single-color spectral references.

  • a.
    Add 4 mL of ice-cold 1x eBioscience permeabilization buffer to a 15 mL tube.
  • b.
    Add 40 drops of CompBeads to the tube.
  • c.
    Mix well on a vortex.
  • d.
    Distribute 100 μL of CompBeads to each well of a V-bottom (conical) 96-well plate.
  • e.
    Add 1 μL of each antibody to each corresponding well.
  • f.
    Incubate overnight in a refrigerator (2−8°C).

Note: Here, we want the fluorochrome on the single-color controls to be in the same condition than the fluorochromes on the cells, as overnight incubation in permeabilization buffer could have an influence on the emission spectra of some fluorochromes.

• 53.Add 100 μL of ice-cold 1x eBioscience permeabilization buffer to the cells and single-color control.
• 54.Centrifuge at 400 × g for 5 min, at 4°C.
• 55.Discard the supernatant.
• 56.Add 200 μL of ice-cold 1x eBioscience permeabilization buffer.
• 57.Centrifuge at 400 × g for 5 min, at 4°C.
• 58.Discard the supernatant.
• 59.Resuspend cell pellets and beads with 200 μL of ice-cold FACS medium.

Data collection

The cells can now be acquired on a spectral flow cytometer and analyzed with FlowJo or equivalent software.

• 60.Collect data on a Sony ID7000 spectral flow cytometer. The spectral flow cytometer needs to be set up accordingly to the manufacturer instruction.
• 61.Acquire the single-color controls to generate the unmixing matrix.
• 62.Acquire the unstained samples to extract the autofluorescence signature.
• 63.Acquire the stained samples.

Note: The current protocol does not include FMO controls (Fluorescence Minus One). However, depending on the biological question, you might include several FMO controls to better define cell positivity for the corresponding markers. Therefore, extra samples from the same tissue and condition are collected and treated as was described for the other samples. Yet, for the FMO control, use an antibody mix excluding the antibody for the marker of interest. (For more information and examples: https://expertcytometry.com/why-use-fmo-controls-multicolor-flow-cytometry-experiment/).

Note: This panel is optimized for the Sony ID7000 equipped with 5 lasers (UV/V/B/YG/R). The use of another spectral analyzer is possible, but compatibility with the panel must be checked and the panel may have to be adapted.

• 64.
  Analyze the samples with FlowJo or analogous software packages to analyze the flow cytometry data (e.g., FCS express).

  Note: The analysis of the collected data should start with a cleaning gating strategy including:

  • a.
    Flow stream stability: the forward scatter parameter should be plotted against time to ensure that the sample run evenly throughout acquisition. If not, events acquired during flow stream instability should be gated out.
  • b.
    Doublet exclusion: area of the forward scatter should be plotted against height of the forward scatter. Cells that follow the diagonal represent the single cells and cells with an increased area relative to the height, represent doublet that should be gated out.
  • c.
    Debris removal: the forward scatter should be plotted against the side scatter to visualize the size and the complexity of the cells. The events with very low forward scatter usually represent cells debris. Be careful to only remove those debris, include the events when in doubt.
  • d.
    Alive leukocytes: finally, draw a gate on the cells that are positives for CD45 and that are not stained by the LIVE/DEAD Fixable Blue.

Expected outcomes

To demonstrate utility and reproducibility, we applied the protocol in a mouse model of type 1 diabetes. The data generated by this protocol can be analyzed with a classical gating strategy. However, this methodology bears limitations, as the gates are defined in a supervised way and their boundaries are not always easy to set.

Here, we performed a high-dimensional analysis by using Uniform Manifold Approximation and Projection (UMAP) using lineage markers. The different populations were identified based on the expression of their lineage markers: CD4 T cells³ (CD3⁺ TCRβ⁺ CD4⁺), CD8 T cells³ (CD3⁺ TCRβ⁺ CD8⁺), double-negative (DN) T cells⁴ (CD3⁺ TCRβ⁺ CD4^- CD8^-), γδ T cells⁵ (CD3⁺ TCRγδ ⁺), NKT cells⁵ (CD3⁺ NKp46⁺), B cells (CD19⁺), NK cells⁵ (CD3^- NKp46⁺), neutrophils⁶ (CD11b⁺ Ly6G⁺), basophils⁷ (CD11b⁺ FcεRIα⁺ c-Kit^-), eosinophils⁸ (SSC^hi CD11b⁺ Siglec-F⁺), macrophages^9,10 (CD11b⁺ F4/80⁺) and dendritic cells¹¹ (F4/80^- CD11c⁺ MHC-II⁺) (Figure 6A). A population was characterized by a lack of expression of any of these markers (Lin^-). These cells might be innate lymphocytes, which could be confirmed by the addition of 2 markers to the current panel: CD127 and Thy1¹² if their identification would be needed to answer the experimental question.

Figure 6.

Example of the data generated with the 40-color panel

(A) Uniform Manifold Approximation and Projection (UMAP) of the different leukocytes identifiable in all samples concatenated.

(B) UMAP split by organ.

(C) Gating strategy to identify cDC1 (CD11b^- CD103⁺), cDC2 (CD11b⁺ CD103^-), (D) islet macrophages (TIM4^- CD206^-) and three subpopulations of exocrine macrophages (TIM4⁺ CD206^-), (TIM4^- CD206⁺), (TIM4⁺ CD206⁺).

(E) UMAP of pancreatic T cells.

(F) Heatmap of the expression of T cell markers defining the different T cell clusters.

The UMAP can then be compared by the experimental condition, like tissue. In the lung, we observed a unique macrophage population (Figure 6B), which are alveolar macrophages as they express SiglecF and CD11c alongside F4/80. This population highlights the heterogeneity of the cells with a myeloid origin and the overlap of the defining markers. Precise gating strategies have been defined in the literature to study specific myeloid populations.^8,10,13 The use of the UMAP may allow to identify the different myeloid populations in an unbiased way without the risk of excluding or mixing populations due to marker overlap.

The leukocyte populations can then be further defined into subpopulations using a classical gating strategy. Dendritic cells can be divided into conventional dendritic cells (cDC) 1 (CD11b^- CD103⁺) and cDC2 (CD11b⁺ CD103^-) (Figure 6C). Macrophages in the pancreas have been shown to have an heterogenous expression of TIM-4 and CD206, defining 4 subpopulations, the islet macrophages (TIM4^- CD206^-) and three subpopulations of exocrine macrophages (TIM4⁺ CD206^-), (TIM4^- CD206⁺), (TIM4⁺ CD206⁺)^9,14 (Figure 6D).

Leukocyte populations with a high heterogeneity, like T cells, can also be analyzed further with high-dimensional analysis. Here, we performed UMAP on pancreatic T cells (Figure 6E) and used a heatmap of the expression of T cell markers to identify the different T cell populations^3,15,16 (Figure 6F).

Mice of different ages and disease stages have been used, which is reflected by some variability in the proportion of leukocytes infiltrating the pancreas, yet an important T cell infiltration can be observed in the pancreas of all mice (Figure 7A). A high variability of pancreatic infiltrating leukocytes is often observed even between mice of the same age,¹⁷ underlying the heterogeneity of disease presentation and response to treatment. In the lung, next to B and T cells, a higher proportion of macrophages and neutrophils is present (Figure 7B). Compared to the other organs, the liver shows the highest proportions of DN T cells, γδ T cells and NK cells (Figure 7C).

Figure 7.

Proportion of different leukocytes

(A) Pancreas.

(B) Lung.

(C) Liver. Each dot represents data from one individual mouse. Data pooled from two independent experiment.

Limitations

This step-by-step protocol allows to analyze most leukocytes present in different organs, yet it has been optimized for the pancreas of NOD/ShiLtJ mice which spontaneously develops type 1 diabetes. We kept the same digestion protocol with collagenase D to keep the experiment simple. In function of the experimental question, the digestion protocol used can be optimized for lung¹³ or liver.¹⁸ Different digestion methods may impact both the yield, and of the different immune populations,^19,20 and cell viability. Some digestion methods may also influence the detection of the surface markers.²¹ As a result, there is no universally optimal protocol.¹⁸ The digestion method used should align with those typically found in literature for the target organ and immune population of interest. Digestion protocols can also be tailored to the specific needs of the experiment. A useful starting point is the work by Burton and colleagues,³ who profiled Treg cells across 30 tissues. From there, different protocols can be compared to identify the one that provides the best results. Importantly, the same digestion protocol should be consistently used across all experiments to ensure comparability.

Inbred strains of laboratory mice have different haplotypes for MHC-II and IgD molecules.^22,23 If another mouse strain will be analyzed with this protocol, the corresponding antibodies will have to be checked for compatibility with the mouse strain and eventually replaced with another clone conjugated to the same fluorochrome.

Troubleshooting

Problem 1

Remaining red blood cells in the samples.

Potential solution

The best solution is to ensure correct perfusion by placing the needle in the left ventricle and keeping it in place during the whole procedure. If the problem persists, increasing the volume to perfused can also improve the removal of residual red blood cell in less vascularized part of the tissue. The last solution is to use a red blood cell lysis buffer which will remove all residual red blood cells but will keep contaminating blood leukocytes in the tissue sample and will increase autofluorescence.

Problem 2

Low cell yield.

Potential solution

• •Ensure that the tissue is cut in tiny pieces (Step 6).
• •Digestion buffer should be pre-heated at 37°C and the enzymes should be added just before use (Step 8).
• •Longer incubation time can reduce the yield.

Problem 3

Low cell viability.

Potential solution

• •Except during digestion, always keep the cells on ice.
• •Longer incubation time during the digestion can increase cell death (Step 10).
• •When layering the cell on top of the Percoll (Step 19), be careful to not mix the 2 phases, which will cause the pellet to be contaminated with cell debris.

Problem 4

Loss of all cells during the staining.

Potential solution

The exposure of the cells to permeabilization buffer before cell fixation (Step 40) will cause permanent loss of the sample. Do not use permeabilization buffer before Step 43.

Problem 5

Abnormal cell positivity for some markers.

Potential solution

This protocol uses anti-mouse CD16/32 Purified antibody and BD BSB+ to limit off-target binding of the antibodies to Fc receptors and Brilliant dye interactions respectively. Cyanine-tandem dye binding to monocyte and macrophages²⁴ can be prevented by adding BD MonoBlock, True-Stain Monocyte Blocker, CellBlox or equivalent reagent to the staining mix, but this may cause a decreased staining for some markers.

A lot of fluorochromes used in this protocol are tandem dyes which are sensible to breakdown, which will cause an increased signal into the parent dye instead of the tandem.

• •Always keep the tandem protected from light and heat.
• •Change reagent vial if tandem breakdown is also observed in the single-color spectral references (Step 52).
• •Cellular enzymes can catalyze tandem breakdown, the use of a tandem stabilizer (BioLegend) can prevent tandem breakdown.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Conny Gysemans (conny.gysemans@kuleuven.be).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Pierre Lemaitre (pierre.lemaitre@kuleuven.be).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The datasets supporting the current protocol have not been deposited in a public repository but are available from the corresponding author on request.

Acknowledgments

We thank Oliver Burton for helpful discussions and his very knowledgeable blog (https://www.colibri-cytometry.com/blog). Furthermore, we thank the KU Leuven Flow Cytometry Core Facility for their help and availability. This work was supported by a Breakthrough T1D (former JDRF) Strategic Research Agreement funding JDRF 2-SRA-2022-1200-S-B and by a KU Leuven small research infrastructure grant KA/20/077. Parts of the visual abstract and of Figure 2 were created with BioRender.com.

Author contributions

P.L., experiment design, panel design, data analysis, and writing – original draft. C.G., writing – review and editing and funding acquisition. C.M., funding acquisition.

Declaration of interests

The authors declare no competing interests.