Protocol for multi-organ isolation of murine leukocytes for flow cytometry

Summary

The immune system comprises several specialized cells across multiple organs. Here, we present a protocol for the isolation of leukocytes from murine blood, spleen, mesenteric lymph nodes, liver, kidney, and skeletal muscle. We describe steps for cell isolation and gradient density separation and provide four antibody panels to identify adaptive immune cells (e.g., CD4⁺ and CD8⁺ T cell subsets) by flow cytometry. This protocol provides a basic framework for phenotyping immune cells from in vivo mouse models.

Graphical abstract

Highlights

• •A streamlined protocol for murine leukocyte isolation from multiple organs
• •Guidelines for multi-organ processing for in vivo mouse models
• •Instructions for multiparameter flow cytometry analyses of immune T lymphocyte subsets

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

The immune system comprises several specialized cells across multiple organs. Here, we present a protocol for the isolation of leukocytes from murine blood, spleen, mesenteric lymph nodes, liver, kidney, and skeletal muscle. We describe steps for cell isolation and gradient density separation and provide four antibody panels to identify adaptive immune cells (e.g., CD4⁺ and CD8⁺ T cell subsets) by flow cytometry. This protocol provides a basic framework for phenotyping immune cells from in vivo mouse models.

Before you begin

Characterization of immune cell populations circulating across different organs can provide valuable insight into how immune cells contribute to homeostatic versus diseased conditions. The immune system encompasses many different populations of leukocytes with specialized roles, which can circulate and be distributed throughout multiple organs to mediate protection against infection, detect neoplastic changes, and maintain immune homeostasis. Key cell populations involved in mediating host defense mechanisms include innate immune cells, such as macrophages, neutrophils and dendritic cells, and adaptive immune T lymphocytes.

T cells are divided into two major populations based on which cluster of differentiation glycoprotein is expressed on their surface: CD4⁺ T cells, classically called helper T cells, and CD8⁺ T cells, known as cytotoxic T cells. Upon exposure to antigen on the surface of antigen presenting cells, naïve CD4⁺ and CD8⁺ T cells become activated, then differentiate into different effector T cell subsets to elicit antigen-specific immune responses.¹ CD4⁺ helper T cell subsets include T helper 1 (Th1), T helper 2 (Th2), T helper 17 (Th17), and T regulatory (Treg) cells. Th1 cells respond to intracellular pathogen infections and have high expression of interferon-γ (IFNγ).² Th1 cell differentiation is induced by the master regulator transcription factor, T-bet, driven by strong signaling from the T cell receptor and the presence of interleukin (IL-) 12 (IL-12).^2,3 In contrast, weak T cell receptor signaling, and the presence of IL-2 and IL-4 activate the GATA3 regulator which drives CD4⁺ T cells to adopt a Th2 cell fate.^2,3,4 Th2 cells mediate immune responses to extracellular pathogens, for example, helminths, and play a role in allergic responses.² Th17 cells respond to bacterial and fungal infections and play pathogenic and non-pathogenic functions in autoimmune diseases such as inflammatory arthritis and multiple sclerosis.^2,4 Th17 cells are characterized by high expression of IL-17A and Th17 cell differentiation is controlled by the transcription factor RORγt, which is activated in the presence of tumor growth factor β and IL-6.^3,4

CD8⁺ T cell subsets include short-lived effector, life-long memory, and exhausted cells. Effector CD8⁺ T cells mediate rapid killing of foreign antigen-expressing cells (e.g., tumor antigens) and cells harboring intracellular pathogens following infection, and can be characterized by high production of pro-inflammatory cytokines, e.g., IFNγ and tumor necrosis factor α (TNFα).⁵ Memory T cells remain long-lived to mediate robust recall responses. In disease contexts of chronic antigen stimulation, such as cancer and chronic infections, CD8⁺ T cells adopt an exhausted state, characterized by high expression of inhibitory receptors, such as PD-1, weakened effector function such as decreased production of pro-inflammatory cytokines.⁶

This protocol focuses on T cell characterization. However, additional panels could be added to enable characterization of innate cell subsets. Appropriate animal ethics must be approved according to institutional guidelines. Experience with multi-parameter (up to 12 color) flow cytometry is necessary. To maintain cell integrity, key equipment and reagents should be prepared in advance of the experiment. The stated time required for each step is for processing of one tissue type, however it is possible to process multiple samples of the same tissue type simultaneously. Each additional tissue must be considered in additional incubation time periods required to process all tissues.

Innovation

The protocol below describes a workflow for the isolation and characterization of leukocytes, primarily T lymphocytes, from the liver, spleen, mesenteric lymph nodes, kidney, skeletal muscle, and peripheral blood of C57BL/6N mice. We then detail procedures for immunophenotyping T cell subsets and in vitro stimulation of T cells for detection of intracellular cytokine production by flow cytometry.

Techniques to isolate immune cells from individual mouse tissues, such as muscle,⁷ secondary lymphoid organs such as the spleen⁸ and lymph nodes,⁹ liver¹⁰ and the kidney¹¹ have been previously described. By adapting and combining previously described techniques of immune cell isolation of various tissues, we present a streamlined workflow for the simultaneous isolation and quantification of T cells across multiple organs of a mouse model for flow cytometry. This protocol facilitates an organism-wide immune cell analysis and enables comparison of organ-specific T cell responses that may impact organ function and host phenotype in health and disease. This protocol can be applied to perform immunophenotyping analysis in multiple disease mouse models for autoimmunity, systemic infection and sepsis, cancer, aging, or diabetes.

Institutional permissions

All experiments for the established protocol were conducted in accordance with approval from the Animal Care Committee (ACC) at the University of Manitoba in full compliance with the Canadian Council on Animal Care, under the Animal Use Protocol (AUP) 21-019.

Percoll preparation

Timing: 30 min

• 1.Prepare fresh stock isotonic Percoll (SIP) by mixing 9 parts Percoll and 1-part 10× (Hank’s Balanced Salt Solution (HBSS) (Table 1). Use SIP to make the different dilutions for density gradient centrifugation i.e., dilute SIP to desired concentration using 1% Fetal Bovine Serum (FBS) HBSS (for isolation of liver lymphocytes) or Roswell Park Memorial Institute (RPMI) (for isolation of kidney and skeletal muscle lymphocytes).
• 2.Prepare 5 mL (per animal) of the following concentrations of SIP: 40% diluted with 1% FBS HBSS, 40% diluted with RPMI, 44% diluted with RPMI, 67% diluted with RPMI, 70% diluted with 1%FBS HBSS, 70% diluted with RPMI, and 80% diluted with RPMI.

Table 1.

Quantities of SIP and diluent required to prepare 5 mL of each required SIP concentration

For use with organ:	Desired concentration of SIP	Volume of 100% SIP	Volume of diluent
Liver	40%	2.00 mL	3.00 mL (1%FBS HBSS)
Liver	70%	3.50 mL	1.50 mL (1%FBS HBSS)
Kidney	44%	2.20 mL	2.80 mL (RPMI)
Kidney	67%	3.35 mL	1.65 mL (RPMI)
Skeletal muscle	40%	2.00 mL	3.00 mL (RPMI)
Skeletal muscle	80%	4.00 mL	1.00 mL (RPMI)

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
APC-Cy7 anti-mouse CD4 (RM4-5) (1:200 dilution)	BioLegend	Cat#100526
Brilliant Violet 421 anti-mouse CD44 (1M7) (1:200 dilution)	BioLegend	Cat#103040
PE anti-mouse CD25 (PC61) (1:200 dilution)	BioLegend	Cat#102008
Alexa Fluor 647 anti-mouse Foxp3 (MF-14) (1:200 dilution)	BioLegend	Cat#126408
PE-Cy7 anti-mouse CD62L (W18021D) (1:200 dilution)	BioLegend	Cat#161213
Alexa Fluor 488 anti-mouse CD11a (2D7) (1:200 dilution)	BioLegend	Cat#162904
PerCPCy5.5 anti-mouse CD8a (53–6.7) (1:200 dilution)	BioLegend	Cat#100734
APC anti-mouse CD44 (IM7) (1:200 dilution)	BioLegend	Cat#103012
APC-Cy7 anti-mouse PD1 (29F.1A12) (1:200 dilution)	BioLegend	Cat#135224
Alexa Fluor 647 anti-mouse IFNγ (XMG1.2) (1:200 dilution)	BioLegend	Cat#505814
PE anti-mouse TNFα (MP6-XT22) (1:200 dilution)	BioLegend	Cat#506306
FITC anti-mouse IL-10 (JES5-16E3) (1:200 dilution)	BioLegend	Cat#505006
PE-Cy7 anti-mouse IL-2 (JES6-5H4) (1:200 dilution)	BioLegend	Cat#503832
Brilliant Violet 605 anti-mouse IL-4 (11B11) (1:200 dilution)	BioLegend	Cat#504126
PE anti-mouse CCR6 (29-2L17) (1:200 dilution)	BioLegend	Cat#129804
Brilliant Violet 650 anti-mouse CD161 (PK136) (1:200 dilution)	BioLegend	Cat#108736
FITC anti-mouse IL-17A (TC11-18H10.1) (1:200 dilution)	BioLegend	Cat#506908
Brilliant Violet 785 anti-mouse CD45 (30-F11) (1:400 dilution)	BioLegend	Cat#103149
Alexa Fluor 700 anti-mouse CD4 (RM4-5) (1:200 dilution)	BioLegend	Cat#100536
Chemicals, peptides, and recombinant proteins
Paraformaldehyde, 4% in phosphate buffered saline (PBS)	Thermo Fisher Scientific	Cat#J61899.AK
Collagenase (type II)	Thermo Fisher Scientific	Cat#17101015
DNase I	STEMCELL Technologies	Cat#07470
Saponin	MiliporeSigma	Cat#47036-50G-F
Sodium azide	MiliporeSigma	Cat#S2002-5G
eBioscience 1× Red Blood Cell (RBC) Lysis Buffer	Thermo Fisher Scientific (Invitrogen)	Cat#00-4333-57
eBioscience Foxp3/Transcription Factor Staining Buffer Set	Thermo Fisher Scientific (Invitrogen)	Cat#00-5523-00
eBioscience Brefeldin A Solution (1000×)	Thermo Fisher Scientific (Invitrogen)	Cat#00-4506-51
eBioscience Cell Stimulation Cocktail (500×)	Thermo Fisher Scientific (Invitrogen)	Cat#00-4970-93
Zombie Aqua Fixable Viability Kit (1:200 dilution)	BioLegend	Cat#423102
Penicillin-Streptomycin-Glutamine (100×)	Thermo Fisher Scientific	Cat#10-378-016
2-Mercaptoethanol (100×)	MiliporeSigma	Cat#ES-007-E
Software and algorithms
FlowJo	BD Life Sciences	https://www.flowjo.com/
GraphPad	Dotmatics	https://www.graphpad.com/
Other
Blood collection syringe with anticoagulant	SAI Infusion Technologies	Sku# HSC500-1-0.03-25
BD Microtainer Tubes with Microgard Closure	BD	Sku# 365974
1× Hank’s Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	Cat#14-025-134
10× Hank’s Balanced Salt Solution (HBSS)	Thermo Fisher Scientific	Cat#14-065-056
Roswell Park Memorial Institute (RPMI) 1640 Medium	Thermo Fisher Scientific	Cat#11-835-055
PBS, pH 7.4	Thermo Fisher Scientific	Cat#10010023
FBS	Corning	Cat#35-015-CV
3 mL sterile syringes	Fisher Scientific	Cat#B309657
60 mm × 15 mm sterile petri dishes	Fisher Scientific	Cat#AS4052
70 μm cell strainers	Fisher Scientific	Cat#08-771-2
Cell scrapers	Fisher Scientific	Cat#08-100-241
LSRFortessa flow cytometer (or equivalent)	BD Biosciences	N/A

Materials and equipment

Digestion Solution (amounts for processing of one mouse, 20 mL per each kidney, liver and muscle tissue sample)

Reagent	Final concentration	Amount
Collagenase (type II)	1 mg/mL	60 mg
DNase I	0.1 mg/mL	6 mg
HBSS	N/A	60 mL
Total	N/A	60 mL

Store at 4°C for no longer than 24 h.

Saponin Permeabilization Buffer (about 8 mL needed per mouse)

Reagent	Final concentration	Amount
Saponin	1 mg/mL (0.1% w/v)	10 mg
Sodium azide	1 mg/mL (0.1% w/v)	10 mg
FBS	1% v/v	0.1 mL
PBS	N/A	10 mL
Total	N/A	10.1 mL

Store at 4°C for no longer than 3 months.

T cell media (TCM)

Reagent	Final concentration	Amount
RPMI	N/A	500 mL
FBS	10% v/v	50 mL
Penicillin, streptomycin, glutamine (100×)	1% v/v	5 mL
2-mercaptoethanol (100×)	0.1% v/v	0.5 mL
Total	N/A	555.5 mL

Store at 4°C for no longer than 3 months.

IMDM (Iscove’s Modified Dulbecco’s Medium) or RPMI can be used as both culture media types support the growth of T cells.

Stop solution

Reagent	Final concentration	Amount
FBS	1% v/v	0.5 mL
HBSS	N/A	50 mL

Step-by-step method details

Tissue collection

Timing: 30 min per mouse

For optimal tissue processing, each tissue is first collected sequentially in a timely manner to maintain tissue integrity.

Note: Store all tissues on ice following collection.

• 1.Collect the blood from a carotid artery bleed into an anticoagulant containing vial (Methods video S1). Note the total volume to be able to calculate cell count per given volume.
• 2.Perfuse the liver through the portal vein using 2 mL of cold HBSS to remove excess blood (Methods video S2). Collect the liver in 5 mL of HBSS supplemented with 1% FBS (Figure 1A) (Methods video S3).
• 3.Collect the spleen in 5 mL of HBSS supplemented with 1% FBS (Figure 1B) (Methods video S3).
• 4.Collect the mesenteric lymph nodes in 5 mL of HBSS supplemented with 1% FBS (Figure 1C) (Methods video S3).
• 5.Collect the two kidneys in 5 mL of RPMI (Figure 1D) (Methods video S3).
• 6.Collect the skeletal muscle in 5 mL of RPMI (Figure 1E) (Methods video S4).

Note: If 5 mL of media is not sufficient to submerge the collected tissue, please utilize a greater volume so that the tissue is fully submerged.

Note: For processing of kidney tissues from both kidneys, it is highly suggested to divide the cell count by two to obtain an estimate for one kidney due to low cell yield. For the optimization of this protocol the following muscles have been used simultaneously: tibialis anterior, soleus, extensor digitorum longus, quadriceps and gastrocnemius, however specific muscle types may be chosen based on the applicability to the study.

Figure 1.

Ex vivo murine tissues

Collected tissues before processing, including (A) liver, (B) spleen, (C) mesenteric lymph nodes, (D) kidneys, and (E) skeletal muscle.

Peripheral blood leukocyte isolation

Timing: 30 min

This section describes how to obtain leukocytes from the blood.

• 7.Centrifuge blood in a microcentrifuge tube at 2250 RCF for 10 min at 20°C–25°C and discard the supernatant.
• 8.Lyse erythrocytes by resuspending the pellet in 500 μL RBC lysis buffer for 4 min at 20°C–25°C.

CRITICAL: Do not overexpose cells to lysis buffer. See troubleshooting, problem 1.

• 9.Stop the reaction by adding 500 μL Stop solution.
• 10.Centrifuge at 2250 RCF for 2 min at 20°C–25°C. Carefully aspirate supernatant and resuspend the cell pellet in 1 mL of HBSS.
• 11.Store cells on ice until cell counting and plating.

Spleen and mesenteric lymph node leukocyte isolation

Timing: 1 h

This section details how to process the spleen and mesenteric lymph nodes to isolate leukocytes.

• 12.
  Spleen leukocyte isolation.

  • a.
    Mash the spleen in a petri dish using the back of a 3 mL syringe plunger until homogenous (Figure 2) (Methods video S5).

    CRITICAL: Ensure the spleen is thoroughly mechanically broken down to maximize the number of cells obtained. See troubleshooting, problem 1.

  • b.
    Strain the suspension using a 70 μm cell strainer and centrifuge at 500 RCF for 5 min at 20°C–25°C to pellet.
  • c.
    Lyse erythrocytes by resuspending the pellet in 1 mL RBC lysis buffer for 4 min.

    CRITICAL: Do not overexpose cells to lysis buffer. See troubleshooting, problem 1.

  • d.
    Stop the reaction by adding 3 mL of Stop solution.
  • e.
    Centrifuge at 500 RCF for 5 min at 20°C–25°C.
  • f.
    Resuspend in 1 mL of PBS.
  • g.
    Store on ice until cell counting and plating.
• 13.
  Mesenteric lymph node leukocyte isolation.

  • a.
    Mash the mesenteric lymph nodes in a petri dish using the back of a 3 mL syringe plunger until homogenous (Figure 3).

    CRITICAL: Ensure the lymph node is thoroughly mechanically broken down to maximize the number of cells obtained. See troubleshooting, problem 1.

  • b.
    Strain the suspension using a 70 μm cell strainer and centrifuge at 500 RCF for 5 min at 20-25°C to pellet.
  • c.
    Lyse erythrocytes by resuspending the pellet in 1 mL RBC lysis buffer for 4 min.

    CRITICAL: Do not overexpose cells to lysis buffer. See troubleshooting, problem 1.

  • d.
    Stop the reaction by adding 3 mL of Stop solution.
  • e.
    Centrifuge at 500 RCF for 5 min at 20-25°C.
  • f.
    Resuspend in 1 mL of PBS.
  • g.
    Store on ice until cell counting and plating.

Figure 2.

Spleen

Spleen (A) pre- and (B) post-processing by manual mashing.

Figure 3.

Mesenteric lymph nodes

Mesenteric lymph nodes (A) pre- and (B) post-processing by manual mashing.

Liver, kidney, and skeletal muscle leukocyte isolation and density gradient purification

Timing: 2 h

This section describes the procedures for processing the liver, kidney, and skeletal muscle to isolate leukocytes.

Note: Store all reagents on ice.

• 14.
  Liver leukocyte isolation.

  • a.
    Transfer the liver into a petri dish with 10 mL digestion solution.
  • b.
    Mince and scrape the liver against the walls of the petri dish using a cell scraper (Methods video S6).
  • c.
    Transfer the mixture into a 15 mL conical tube with 10 mL digestion solution (Figure 4A).
  • d.
    Incubate the mixture at 37°C for 20 min.
  • e.
    Transfer the contents back into a petri dish through a 70 μm cell strainer (Figure 4B).
  • f.
    Using the rubber end of a 3 mL syringe plunger, mash the mixture (Methods video S7), and strain the mixture using a new 70 μm cell strainer into a fresh 50 mL conical tube.

    CRITICAL: Ensure the liver is thoroughly mechanically broken down to maximize the number of cells obtained. See troubleshooting, problem 1.

  • g.
    Centrifuge the tube at 350 RCF or 10 min at 20-25°C. Wash the pellet using cold HBSS.
  • h.
    Lyse erythrocytes by resuspending the pellet in 3 mL RBC lysis buffer for 3 min.

    CRITICAL: Do not overexpose cells to lysis buffer. See troubleshooting, problem 1.

  • i.
    Stop the reaction by adding 10 mL of 1% FBS HBSS.
  • j.
    Centrifuge at 350 RCF for 10 min at 20°C–25°C.
• 15.
  Kidney leukocyte isolation.

  • a.
    Transfer the kidney into a petri dish with 10 mL digestion solution (Figure 5A).
  • b.
    Mince and scrape the kidney using a cell scraper.
  • c.
    Transfer the mixture into a 15 mL conical tube with 10 mL digestion solution.
  • d.
    Incubate the mixture at 37°C for 20 min.
  • e.
    Transfer the contents back into a petri dish through a 70 μm cell strainer (Figure 5B).
  • f.
    Using the rubber end of a 3 mL syringe plunger, mash the mixture, and strain the mixture using a new 70 μm cell strainer into a fresh 50 mL conical tube.

    CRITICAL: Ensure the kidney is thoroughly mechanically broken down to maximize the number of cells obtained. See troubleshooting, problem 1.

  • g.
    Centrifuge the tube at 350 RCF for 10 min at 20°C–25°C. Wash the pellet using cold RPMI.
  • h.
    Lyse erythrocytes by resuspending the pellet in 3 mL RBC lysis buffer for 3 min.

    CRITICAL: Do not overexpose cells to lysis buffer. See troubleshooting, problem 1.

  • i.
    Stop the reaction by adding 10 mL of Stop solution.
  • j.
    Centrifuge at 350 RCF for 10 min at 20°C–25°C.
• 16.
  Skeletal muscle leukocyte isolation.

  • a.
    Transfer the skeletal muscle into a petri dish with 10 mL digestion solution (Figure 6A).
  • b.
    Mince and scrape the muscle using a cell scraper.
  • c.
    Transfer the mixture into a 15 mL conical tube with 10 mL digestion solution.
  • d.
    Incubate the mixture at 37°C for 20 min.
  • e.
    Transfer the contents back into a petri dish through a 70 μm cell strainer (Figure 6B).
  • f.
    Using the rubber end of a 3 mL syringe plunger, mash the mixture, and strain the mixture using a new 70 μm cell strainer into a fresh 50 mL conical tube.

    CRITICAL: Ensure the muscle is thoroughly mechanically broken down to maximize the number of cells obtained. See troubleshooting, problem 1.

  • g.
    Centrifuge at 350 RCF for 10 min at 20-25°C. Wash the pellet using cold RPMI.
  • h.
    Lyse erythrocytes by resuspending the pellet in 3 mL RBC lysis buffer for 3 min.

    CRITICAL: Do not overexpose cells to lysis buffer.

  • i.
    Stop the reaction by adding 10 mL of Stop solution.
  • j.
    Centrifuge at 350 RCF for 10 min at 20°C–25°C.
• 17.
  Density Gradient preparation. Different leukocyte populations can be isolated based on their density.

  • a.
    Liver gradient preparation.

    • i.
      Resuspend the liver cell pellet in 5 mL of 40% Percoll.
    • ii.
      Using the gravity setting on the serological pipette gun carefully pipette the 40% Percoll mixture on top of 5 mL 70% Percoll to create the layered gradient (Methods video S8).
  • b.
    Kidney gradient preparation.

    • i.
      Resuspend the kidney cell pellet in 5 mL of 44% Percoll.
    • ii.
      Carefully pipette the 44% Percoll mixture on top of 5 mL 67% Percoll to create the layered gradient.
  • c.
    Skeletal muscle gradient preparation.

    • i.
      Resuspend the muscle cell pellet in 5 mL of 40% Percoll.
    • ii.
      Carefully pipette the 40% Percoll mixture on top of 5 mL 80% Percoll to create the layered gradient.
• 18.
  Density gradient centrifugation.

  • a.
    Using the lowest acceleration setting and no break, centrifuge the tubes containing gradients at 900 RCFor 20 min at 4°C.

    CRITICAL: Do not shake or bump the tubes as it could disturb the gradients. See troubleshooting, problem 2.

  • b.
    Carefully collect the lymphocyte interfaces (Figure 7) (Methods video S9) into fresh 15 mL conical tubes and wash with 10 mL cold 1% FBS HBSS (liver) or RPMI (kidney and skeletal muscle). Centrifuge cells at 900 RCF for 5 min at 20-25°C.

    Note: See Figure 7 for example lymphocyte interfaces.

  • c.
    Resuspend the cell pellet in 1 mL PBS for cell counting and staining for flow cytometry.

Figure 4.

Liver

Liver (A) pre- and (B) post-digestion.

Figure 5.

Kidneys

Kidneys (A) pre- and (B) post-digestion.

Figure 6.

Skeletal muscle

Skeletal muscle (A) pre- and (B) post-digestion.

Figure 7.

Tissue percoll density gradients

Percoll density gradients of (A) liver, (B) kidney, and (C) skeletal muscle. The arrows indicate the lymphocyte interface.

Staining of murine leukocytes to identify T cell populations by flow cytometry

Timing: 6 h

This section describes leukocyte sample preparation for surface and intracellular protein antibody staining for flow cytometry.

Note: Store all reagents on ice.

• 19.
  Cell counting and 96-well plate preparation.

  • a.
    Using preferred method (hemocytometer, flow cytometry, automated cell counter, etc.) count the total cells acquired from each organ and record as # cells/mL PBS.
  • b.
    Label 4 96-well plates A-D, one plate for each flow cytometry panel.
  • c.
    In each plate, allocate and label one well per organ, per animal, Fluorescent Minus One (FMO) control wells, and unstained control well.
  • d.
    For organs with yield higher than 4×10⁶ cells, seed the wells with 1×10⁶cells.

    Note: Calculate the volume of PBS-suspended cells required to seed 1×10⁶ cells per well in μL as follows:

     x = volume cell concentration needed for 1×10⁶ cells per well.
     For example, if the total cell count/volume of PBS (1000 μL) = 25×10⁶ cells per 1 mL (or 1000 μL).
     x = (1×10⁶ cells)/(25×10⁶ cells/1 mL)= 0.04 mL or 40 μL.
  • e.
    For organs with lower yields, divide the cells evenly into the 4 panels (250 μL of the PBS-suspended cells).
  • f.
    Centrifuge the cells at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • g.
    Resuspend the cells in 200 μL TCM.
• 20.
  Antibody master mix preparation.

  • a.
    Prepare panel A cell surface stain antibody cocktail (Table 2).

    • i.
      Multiply the number of 96-wells used plus one extra well for overage for panel A by 100 μL to calculate the volume of PBS required.

      Note: For example, if there are 24 wells used + 1 extra well: 25 wells x 100 μL PBS = 2500 μL of PBS.

    • ii.
      Multiply the number of 96-wells used for panel A by 0.5 μL (0.5 μL is a 1 in 200 dilution of the antibody in 100 μL PBS total per sample) to calculate the volume needed of each antibody.

      Note: For example, if there are 24 wells used + 1 extra well for overage: 25 wells, use 25 × 0.5 μL = 12.5 μL of each antibody.

      CRITICAL: If the flow cytometry signal for a given fluorophore is not optimal, a different ratio of antibody to PBS may be used. Please adjust the volume of antibody used accordingly (1 μL per well for 1:100, 0.33 μL per well for 1:300, 0.25 μL per well for 1:400, etc.) See troubleshooting, problem 3.

    • iii.
      Add the following antibodies to PBS: CD45-Brilliant Violet 785, CD4-APCCy7, CD44-Brilliant Violet 421, CD25-PE, CD62L-PECy7, and CD11a-Alexa Fluor 488. Use the volume of PBS calculated in step i and the volume of antibodies calculated in step ii.
  • b.
    Prepare panel B cell surface stain antibody cocktail (Table 2).

    • i.
      Calculate the volume of PBS required as in step 20a, i.
    • ii.
      Calculate the volume needed of each antibody as in step 20a, ii.
    • iii.
      Add the following antibodies to PBS: CD45-Brilliant Violet 785, CD4-Alexa Fluor 700, CD8a-PerCPCy5.5, CD44-APC, PD-1-APCCy7, CD11a-Alexa Fluor 488. Use the volume of PBS calculated in step i and the volume of antibodies calculated in step ii.
  • c.
    Prepare panel C cell surface stain antibody cocktail (Table 2).

    • i.
      Calculate the volume of PBS required as in step 20a, i.
    • ii.
      Calculate the volume needed of each antibody as in step 20a, ii.
    • iii.
      Add the following antibodies to PBS: CD45-Brilliant Violet 785, CD4-APCCy7, CD8a-PerCPCy5.5, CD44-Brilliant Violet 421. Use the volume of PBS calculated in step i and the volume of antibodies calculated in step ii.
  • d.
    Prepare panel D cell surface stain antibody cocktail (Table 2).

    • i.
      Calculate the volume of PBS required as in step 20a, i.
    • ii.
      Calculate the volume needed of each antibody as in step 20a, ii.
    • iii.
      Add the following antibodies to PBS: CD45-Brilliant Violet 785, CD4-APCCy7, CD44-Brilliant Violet 421, CCR6-PE, CD161-Brilliant Violet 650. Use the volume of PBS calculated in step i and the volume of antibodies calculated in step ii.
  • e.
    For each panel, prepare Fluorescent Minus One (FMO) control stains. FMO controls are cell samples that are stained with all antibodies minus one antibody in the panel. Use the same antibody dilution as in 20a, ii; 20b, ii; 20c, ii; and 20d ii for panels A-D. Include an unstained negative control well which contains cells and PBS only in a total volume of 100 μL. It is recommended to utilize spleen cells for the FMO controls and unstained negative control due to their relative abundance compared to other organs.

    Note: Panel A is designed to characterize regulatory CD4⁺ T cells.

    Note: Panel B is designed to characterize naïve CD4⁺ T cells, activated CD4⁺ T cells, naïve CD8⁺ T cells, activated CD8⁺ T cells, and exhausted CD8⁺ T cells.

    Note: Panel C is designed to characterize Th1 cells, Th2 cells, and effector CD8⁺ T cells.

    Note: Panel D is designed to characterize Th17 cells.

    Note:Table 3 describes the cell phenotypes used to identify the cell populations in their respective panels.

    Note: Further optimization of antibody concentrations may be required. See troubleshooting, problem 3.

    Note: For each multicolor antibody panel, it is highly suggested to include FMO control stains, and an unstained cells negative control, for optimal gating strategies to distinguish between positive and negative cell populations and background fluorescence. Inclusion of FMO controls enables the user to set the appropriate gate for positive and negative cell populations during downstream flow cytometry data analysis (see Table 4 as an example).

• 21.
  Cell stimulation for intracellular protein staining.

  Note: This intracellular protein staining protocol uses Brefeldin A (BFA) solution 1000× and Cell Stimulation Cocktail 500×.

  • a.
    Add 0.1 μL BFA to each panel C and D well to stop intracellular protein transport.
  • b.
    To stimulate cells for intracellular cytokine detection, add 2 μL of PMA/ionomycin cocktail (1-part eBioscience Cell Stimulation Cocktail 500× mixed with 9 parts TCM) into each panel C and D well. Include an extra well with 0.1 μL BFA only to be used as an unstimulated control comparison. This unstimulated control well does not contain the Cell Stimulation Cocktail.
  • c.
    Incubate cells for 4 h at 37°C.
• 22.
  Cell surface protein staining protocol (All panels).

  • a.
    Viability staining.

    • i.
      Calculate the volume of PBS needed to stain all cells from all 4 panels, 100 μL per well. For example, if each of the 4 panels has 24 wells, 9600 μL of PBS are needed.
    • ii.
      Calculate the volume of zombie aqua dye needed to stain all cells from all 4 panels, 1 μL per well. For example, if each of the 4 panels has 24 wells, 96 μL of zombie aqua dye are needed.
    • iii.
      Reconstitute zombie aqua dye with DMSO provided in the kit, 100 μL per vial. Reconstitute a number of vials sufficient to stain all wells in all panels (one vial holds 100 μL, thus, for example, if 96 wells are being stained, only one vial is required).
    • iv.
      Add the volume of zombie aqua dye calculated in step ii to the volume of PBS calculated in step i to make the zombie aqua cocktail.
    • v.
      Centrifuge cells at 900 RCF for 5 min at 20-25°C. Remove supernatant.
    • vi.
      Resuspend cells in 100 μL of the zombie aqua cocktail.
    • vii.
      Stain cells with zombie aqua dye for 20 min in 4°C the dark.
    • viii.
      Wash the cells with 200 μL of PBS. Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • b.
    Resuspend cells from each panel in 100 μL of their respective surface stain antibody cocktail and stain for 15 min at 4°C in the dark.
  • c.
    Wash the cells with 200 μL of PBS. Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant. For panels B, C and D proceed to step 23. For panel A, skip steps 23 and 24, proceed to step 25.
• 23.
  Cell fixation (panels B, C and D).

  • a.
    Resuspend the cells in 100 μL 4% paraformaldehyde.
  • b.
    Fix the cells in paraformaldehyde for 10 min at 20-25°C in the dark.
  • c.
    Wash the cells with 200 μL of PBS. Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • d.
    For panel B, resuspend the cells in 200 μL PBS and store at 4°C for flow cytometry acquisition. For panels C and D proceed to step 24.
• 24.
  Intracellular staining (panels C and D).

  • a.
    Prepare panel C intracellular staining antibody cocktail.

    • i.
      Multiply the number of 96-wells used plus one extra well for overage for panel C by 100 μL to calculate the volume of Saponin Permeabilization Buffer required. For example, if there are 24 wells used + 1 extra well, use 2500 μL of Saponin Permeabilization Buffer (25 wells x 100 μL).
    • ii.
      Multiply the number of 96-wells used for panel C by 0.5 μL to calculate the volume needed of each antibody. For example, if there are 24 wells used + 1 extra well for overage, use 12.5 μL of each antibody (0.5 μL x 25 wells).

      CRITICAL: If the flow cytometry signal for a given fluorophore is not optimal a different ratio of antibody to Saponin Permeabilization Buffer may be used. Please adjust the volume of antibody used accordingly (1 μL per well for 1:100, 0.33 μL per well for 1:300, 0.25 μL per well for 1:400, etc.) See troubleshooting, problem 3.

    • iii.
      Add the following antibodies to the Saponin Permeabilization Buffer: IFNγ-Alexa Fluor 647, TNFα-PE, IL-10-FITC, IL-2-PECy7, IL-4-Brilliant Violet. Use the volume of Saponin Permeabilization Buffer calculated in step i and the volume of antibodies calculated in step ii.
  • b.
    Prepare panel D intracellular stain antibody cocktail.

    • i.
      Multiply the number of 96-wells used plus one extra well for overage for panel D by 100 μL to calculate the volume of Saponin Permeabilization Buffer required, as in 24a, i.
    • ii.
      Multiply the number of 96-wells used + 1 extra well for overage for panel D by 0.5 μL to calculate the volume needed of the IL-17A-FITC antibody.

      CRITICAL: If the flow cytometry signal for a given fluorophore is not optimal a different ratio of antibody to Saponin Permeabilization Buffer may be used. Please adjust the volume of antibody used accordingly (1 μL per well for 1:100, 0.33 μL per well for 1:300, 0.25 μL per well for 1:400, etc.) See troubleshooting, problem 3.

    • iii.
      Add the volume of IL-17A-FITC antibody calculated in step i to the volume of Saponin Permeabilization Buffer calculated in step ii.
  • c.
    Wash the cells with 200 μL of Saponin Permeabilization Buffer. Centrifuge at 900 RCF for 5 min. Remove the supernatant.
  • d.
    Add 100 μL of the panel C intracellular stain antibody cocktail to the panel C wells. Add 100 μL of the panel D intracellular stain antibody cocktail to the panel D wells.
  • e.
    Stain cells for 30 min, at 20-25°C in the dark.
  • f.
    Wash the cells with 100 μL Saponin Permeabilization Buffer. Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • g.
    Repeat step f.
  • h.
    Wash the cells with 200 μL PBS. Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • i.
    Resuspend the cells in 200 μL PBS and store in 4°C for flow cytometry acquisition.
• 25.
  Transcription Factor staining protocol (panel A).

  Note: This staining protocol uses the eBioscience FoxP3/Transcription Factor Staining Buffer Set.

  • a.
    Resuspend the previously surface stained cells of panel A in 200 μL FoxP3 Fixation/Permeabilization (fix/perm) solution (1-part FoxP3 Fixation/Permeabilization Concentrate mixed with 3 parts FoxP3 Fixation/Permeabilization Diluent, eBioscience™ Foxp3/Transcription Factor Staining Buffer Set) for an hour at 4°C, in the dark.
  • b.
    Centrifuge at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • c.
    Wash cells using 200 μL 1× FoxP3 kit permeabilization buffer as prepared in a. Centrifuge the cells at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • d.
    Repeat step c.
  • e.
    Prepare panel A FoxP3 staining antibody cocktail.

    • i.
      Multiply the number of 96-wells used plus one extra well for overage for panel A by 100 μL to calculate the volume of 1× FoxP3 kit permeabilization buffer required. For example, if there are 24 wells used + 1 extra well for overage, use 2500 μL of 1× FoxP3 kit permeabilization buffer.
    • ii.
      Multiply the number of 96-wells used + 1 extra well for overage for panel A by 0.5 μL to calculate the volume needed of the FoxP3-Alexa Fluor 647 antibody. For example, use 12.5 μL of the antibody for 25 (24 wells used + 1 extra well for overage).

      CRITICAL: If the flow cytometry signal for a given fluorophore is not optimal, a different ratio of antibody to 1× FoxP3 kit permeabilization buffer may be used. Please adjust the volume of antibody used accordingly (1 μL per well for 1:100, 0.33 μL per well for 1:300, 0.25 μL per well for 1:400, etc.) See troubleshooting, problem 3.

    • iii.
      Add the volume of FoxP3-Alexa Fluor 647 antibody calculated in step i to the volume of 1× FoxP3 kit permeabilization buffer calculated in step ii.
  • f.
    Stain cells with FoxP3 stain antibody cocktail for 30 min at 20-25°C in the dark.
  • g.
    Stop the staining by washing with 100 μL of 1× FoxP3 kit permeabilization buffer. Centrifuge the cells at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • h.
    Repeat step g.
  • i.
    Wash the cells with 200 μL PBS. Centrifuge the cells at 900 RCF for 5 min at 20-25°C. Remove the supernatant.
  • j.
    Resuspend the cells in 200 μL PBS and store at 4°C for flow cytometry acquisition.

Table 2.

Description of antibodies used in flow cytometry panels, related to Step 20

Marker	Clone	Fluorophore	Laser	Catalog #	Company	Staining protocol
PANEL A
CD45	30-F11	BV 785	Violet	103149	Biolegend	Surface
CD4	RM4-5	APC-Cy7	Red	100526	Biolegend	Surface
CD44	1M7	BV421	Violet	103040	Biolegend	Surface
CD25	PC61	PE	Green	102008	Biolegend	Surface
Foxp3	MF-14	AF647	Red	126408	Biolegend	Foxp3 fix/perm
CD62L	MEL-14	PE-Cy7	Green	104418	Biolegend	Surface
CD11a	2d7	AF488	Blue	162904	Biolegend	Surface
Viability Dye	N/A	Zombie Aqua (BV510)	Violet	423102	Biolegend	Viability staining
PANEL B
CD45	30-F11	BV 785	Violet	103149	Biolegend	Surface
CD4	RM4-5	AF700	Violet	100536	Biolegend	Surface
CD8a	53–6.7	PerCPCy5.5	Blue	100734	Biolegend	Surface
CD62L	MEL-14	PE-Cy7	Green	104418	Biolegend	Surface
CD44	IM7	APC	Red	103012	Biolegend	Surface
PD1	29F.1A12	APC-Cy7	Red	135224	Biolegend	Surface
CD11a	2d7	AF488	Blue	162904	Biolegend	Surface
Viability Dye	N/A	Zombie Aqua (BV510)	Violet	423102	Biolegend	Viability staining
PANEL C
CD45	30-F11	BV 785	Violet	103149	Biolegend	Surface
CD4	RM4-5	APC-Cy7	Red	100526	Biolegend	Surface
IFNγ	XMG1.2	AF647	Red	505814	Biolegend	4% PFA fix/perm
TNFα	MP6-XT22	PE	Green	506306	Biolegend	4% PFA fix/perm
CD8	53–6.7	PerCPCy5.5	Blue	100734	Biolegend	Surface
IL-10	JES5-16E3	FITC	Blue	505006	Biolegend	4% PFA fix/perm
IL-2	JES6-5H4	PE-Cy5	Green	503824	Biolegend	4% PFA fix/perm
IL-4	11B11	BV605	Violet	504126	Biolegend	4% PFA fix/perm
CD44	1M7	BV421	Violet	103040	Biolegend	Surface
Viability Dye	N/A	Zombie Aqua (BV510)	Violet	423102	Biolegend	Viability staining
PANEL D
CD45	30-F11	BV 785	Violet	103149	Biolegend	Surface
CD4	RM4-5	APC-Cy7	Red	100526	Biolegend	Surface
CCR6	29-2L17	PE	Green	129804	Biolegend	Surface
CD161	PK136	BV650	Violet	108736	Biolegend	Surface
IL-17A	TC11-18H10.1	FITC	Blue	506908	Biolegend	4% PFA fix/perm
CD44	1M7	BV421	Violet	103040	Biolegend	Surface
Viability Dye	N/A	Zombie Aqua (BV510)	Violet	423102	Biolegend	Viability staining

Table 3.

Summary of immune cell phenotypes, related to Step 20

Cell type	Phenotype used to identify cell type	Panel used to identify cell type
Naïve CD4+ T cells	CD4+CD62LhiCD44lo	B
Activated CD4+ T cells	CD4+CD62LloCD44hiCD11ahi	B
Regulatory T cells	CD4+CD44+CD25+FoxP3+	A
T helper 1 cells	CD4+CD44+IFNγ+TNFα+IL-2+	C
T helper 2 cells	CD4+CD44+IL-4+IL-10+	C
T helper 17 cells	CD4+CD44+CCR6+CD161+IL-17A+	D
Naïve CD8+ T cells	CD8+CD62LhiCD44lo	B
Activated CD8+ T cells	CD8+CD62LloCD44hiCD11ahi	B
Exhausted CD8+ T cells	CD8+CD44hiCD11ahiPD-1hi	B
Effector CD8+ T cells	CD8+CD44+IFNγ+TNFα	C

Table 4.

Example of 96-well plate format for Panel A surface stains including unlabeled empty wells, related to Step 19

	1	2	3	4	5	6	7	8	9	10	11	12
A	Mouse 1 Blood	Mouse 1 Spleen	Mouse 1 MLN	Mouse 1 Liver	Mouse 1 Kidney	Mouse 1 SM
B
C	Mouse 2 Blood	Mouse 2 Spleen	Mouse 2 MLN	Mouse 2 Liver	Mouse 2 Kidney	Mouse 2 SM
D
E	Mouse 3 Blood	Mouse 3 Spleen	Mouse 3 MLN	Mouse 3 Liver	Mouse 3 Kidney	Mouse 3 SM
F
G	FMO CD45-Brilliant Violet 785	FMO CD4-APCCy7	FMO CD44-Brilliant Violet 421	FMO CD25-PE	FMO CD62L-PECy7Violet 421	FMO CD62L-PECy7	FMO CD11a-Alexa Fluor 488	FMO CD11a-Alexa Fluor 488	Unstained cells
H

Flow cytometry data acquisition and data analysis

Timing: 4 h

Flow cytometry data can be acquired using a multiparameter flow cytometer and supporting data analysis software, as described in this section.

• 26.Acquire flow cytometry data using a LSRFortessa flow cytometer or equivalent, following instrument standard operating methods.
• 27.Data analysis is performed using FlowJo v10 software. Detailed instructions for flow cytometry data analysis using FlowJo is made freely available to all users in FlowJo Basic Tutorial Manuals.

Note: For sample acquisition, the flow speed was 2 μL/sec speed on the high throughput sampler (HTS), which is twice as fast as the fast flow speed in tube mode.

Expected outcomes

Following data acquisition on a flow cytometer, the data must be gated accordingly using FlowJo software in order to identify the T cell populations of interest. Figures 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17 illustrate the appropriate gating strategies for identifying the populations of each cell subtype.

Figure 8.

Gating strategy for panel A for all tissues

Lymphocytes are first gated on the side (SSC) and forward scatter (FSC) plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the blood, liver, spleen, kidneys, mesenteric lymph nodes, and skeletal muscle. Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ T cell frequencies are shown.

Figure 9.

Gating strategy for panel B for all tissues

Lymphocytes are first gated on the side (SSC) and forward scatter (FSC) plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the blood, liver, spleen, kidneys, mesenteric lymph nodes, and skeletal muscle. Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ and CD8⁺ T cell frequencies are shown.

Figure 10.

Gating strategy for panel C for all tissues

Lymphocytes are first gated on the side (SSC) and forward scatter (FSC) plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the blood, liver, spleen, kidneys, mesenteric lymph nodes, and skeletal muscle. Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ and CD8⁺ T cell frequencies are shown.

Figure 11.

Gating strategy for panel D for all tissues

Lymphocytes are first gated on the side (SSC) and forward scatter (FSC) plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the blood, liver, spleen, kidneys, mesenteric lymph nodes, and skeletal muscle. Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ T cell frequencies are shown.

Figure 12.

Gating strategy for panel A to identify CD4⁺ regulatory T cells

Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4+ T cells positive for CD44, CD25 and FoxP3 expression are identified as regulatory T cells. Fluorescence Minus One (FMO) gating controls are shown.

Figure 13.

Gating strategy for panel B to identify naïve and activated CD4⁺ or CD8⁺ T cells

(A) Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ or CD8⁺ T cells are then gated on expression of CD62L, with naïve CD4⁺ or CD8⁺ T cells exhibiting a CD44^loCD62L^hi profile and conventionally activated CD4⁺ or CD8⁺ T cells T cells exhibiting a CD44^hiCD62L^lo profile. Resting memory phenotype T cells have been also described as CD44^hi. Representative plots of (B) CD11a+ splenic CD4⁺ and CD8⁺ T cells, and (C) PD-1+ CD8+ T cells in the skeletal muscle. Fluorescence Minus One (FMO) gating controls are shown.

Figure 14.

Gating strategy for panel C to identify CD4⁺ Th1 cells

Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ T cells positive for CD44 are gated on TNFα+ cells, followed by IL-2 and IFNγ. Th1 cells exhibit a triple positive IL-2+IFNγ+TNFα+ profile. Fluorescence Minus One (FMO) and unstimulated sample gating controls are shown.

Figure 15.

Gating strategy for panel C to identify CD4⁺ Th2 cells

Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ T cells positive for CD44 are gated on IL-4 and IL-10 positive cells. Th2 cells exhibit an IL-4+IL-10+ profile. Fluorescence Minus One (FMO) and unstimulated sample gating controls are shown.

Figure 16.

Gating strategy for panel C to identify effector CD8⁺ T cells

Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD8⁺ T cells positive for CD44 are gated on IFNγ versus TNFα, where the double positive gate is used to identify the IFNγ+TNFα+CD44+ effector CD8⁺ T cells. Fluorescence Minus One (FMO) and unstimulated sample gating controls are shown.

Figure 17.

Gating strategy for panel D to identify CD4⁺ Th17 cells

Lymphocytes are gated on the side (SSC) and forward (FSC) scatter plot, followed by a gate to identify singlets. Gated on singlets, CD45+ cells confirm the presence of leukocytes in the tissue (spleen is shown). Gated on CD45+ cells, live versus dead cells are distinguished using Zombie Aqua. Gated on live cells, CD4⁺ T cells are then gated on CD44 expression. CD4⁺CD44^hi cells are gated for CCR6 and CD161 and the double positive cells are then gated on IL-17A to identify IL-17A+CCR6+CD161+ Th17 cells. Fluorescence Minus One (FMO) and unstimulated sample gating controls are shown.

Once the different T cell populations have been identified, the absolute cell count of each leukocyte population can be calculated by multiplying the percentage of cells in a given population by the volume of the sample acquired and the events per volume measurement. The resulting absolute cell counts can then be used to examine how immune populations change across different variables and conditions. For example, Figure 18 compares cell counts between healthy male (n=2) and female (n=3) mice. Since the immune system is highly individualized, it is important to note the variability in cell numbers, even between individuals under the same conditions. Similarly, less prominent cell types may not be detectable in every sample. Therefore, appropriate sample sizes should be chosen to derive statistically significant conclusions from each experiment.

Note: The calculation of absolute cell count based upon volume might give incorrect results if the instrument is not in optimal condition.

Figure 18.

Example cell counts obtained from healthy male (n=2) and female (n=3) C57BL/6N mice following the protocol

Note not all cell types were detected in each organ from each mouse.

Limitations

This protocol does not differentiate between tissue resident leukocytes and circulating subsets within the vascular system of the organ. Recent studies suggest that tissue resident cells have different transcriptional, proteomic, and cell-surface profiles compared to their circulating counterparts.^12,13,14,15 If those distinctions are relevant to the study, we suggest adding panels with antibody markers suited to specifically identifying tissue resident cells. As the organs are homogenized and digested, it is likely that cells found within the blood vessels will also be collected; these comprise a varying proportion of the total leukocytes for each organ, depending on the relative blood content, and are always a minority fraction. If this is undesirable for a given application, we suggest perfusing the organ to minimize the amount of blood present, as has been done for the liver given the high ratio of blood to tissue.

Isolating immune cells from muscle tissue can be difficult and produce low yields. Similarly, another limitation is the low cell number of lymphocytes in the kidneys observed under homeostatic conditions.¹⁶ An alternative method for immune cell isolation is the use of CD45 magnetic bead-based enrichment which can be used to increase cell yields.

Troubleshooting

Problem 1

Cell counts lower than expected.

Potential solution

Ensure organs are thoroughly broken down mechanically as insufficient breakdown would lead to cells being filtered out with the tissue fragments. Increasing the length of the digestion step to obtain the maximum number of cells may be necessary for the liver, kidney and muscle tissues. Do not overexpose cells to lysis buffer as it could damage the integrity of desirable cells.

Problem 2

Percoll gradient cell interface not visible.

Potential solution

If the interface is not formed properly during pipetting, or if the tubes are shaken or bumped (including before the centrifugation), the Percoll interface will dissipate and the gradient will not produce a visible lymphocyte layer. If this occurs or the lymphocyte interface is not readily apparent, repeat the centrifugation step.

Problem 3

Antibody signal is too weak or too strong following data acquisition.

Potential solution

While the provided concentration of antibodies was tested and optimized, further titration of antibodies might be needed to achieve optimal signal strength for detection of immune cells from different disease models. This is particularly important when using alternate fluorophores or antibody clones.

Problem 4

Skeletal muscle suspension doesn’t pass easily through the 70 μm filter.

Potential solution

The muscle tissue suspension can be viscous following digestion. If the filter is not straining the solution easily, pipette the tissue suspension in fractions to avoid overloading the filter. Stirring the suspension within the filter is also suggested (the plunger used in step 16 f may be used for this purpose).

Problem 5

Low detection of intracellular cytokine protein expression after cell stimulation for 4 h.

Potential solution

If the positive shift for intracellular cytokine detection in the stimulated sample is minimal compared to the unstimulated sample control, a longer incubation time of cell stimulation (e.g., at least 6 h) can be performed to increase the detection of intracellular cytokine expression.

Resource availability

Lead contact

Requests for resources and further information should be directed to the lead contact, Janilyn Arsenio (janilyn.arsenio@umanitoba.ca).

Technical contact

Technical questions should be directed to the technical contact, Janilyn Arsenio (janilyn.arsenio@umanitoba.ca).

Materials availability

This protocol did not generate new reagents.

Data and code availability

• •Data questions should be directed to Janilyn Arsenio.
• •This protocol did not generate new code.

Acknowledgments

We would like to acknowledge the Central Animal Care Services staff at the University of Manitoba, the Rady Faculty of Health Sciences Department of Immunology Flow Cytometry Core at the University of Manitoba, Winnipeg, Manitoba, Canada. This work was supported by the Canada Research Chairs (CRC) Program (J.A. is a Tier 2 CRC in Systems Biology of Chronic Inflammation), Canadian Foundation for Innovation John R. Evans Leaders Fund (CFI-JELF) Project #37784 (awarded to J.A.), and the Manitoba Medical Services Foundation (A.A.M. holds the Dr. F.W. DuVal and John Henson Clinical Research Professorship).

Author contributions

Conceptualization, A.A.M. and J.A.; methodology, P.J.L., A.M., A.A.M., and J.A.; formal analysis, P.J.L., A.M., and J.A.; investigation, P.J.L., D.S., and A.M.; resources, A.A.M. and J.A.; writing – original draft, P.J.L. and A.M.; writing – review and editing, A.M., D.S., A.A.M., and J.A.; visualization, A.M. and J.A.; supervision, A.A.M. and J.A.; project administration, A.A.M. and J.A.; funding acquisition, A.A.M. and J.A.

Declaration of interests

The authors declare no competing interests.