Protocol to examine immune subpopulations in murine conjunctiva and lacrimal gland using flow cytometry

Summary

Here, we present a protocol for the examination of immune cells in the murine conjunctiva and lacrimal gland using flow cytometry. We describe steps for dissection, preparation of high-quality single-cell suspensions, utilization of comprehensive staining panels, and optimization of flow cytometry voltage. We then detail procedures for compensation adjustments and the implementation of effective gating strategies.

For complete details on the use and execution of this protocol, please refer to Ma et al.¹

Graphical abstract

Highlights

• •Dissection of murine conjunctiva and lacrimal gland
• •Preparation of single-cell suspension from murine conjunctiva and lacrimal gland
• •Antibody staining for effector T cells and ILCs
• •Flow cytometry gating strategy for effector T cell and ILCs

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

Here, we present a protocol for the examination of immune cells in the murine conjunctiva and lacrimal gland using flow cytometry. We describe steps for dissection, preparation of high-quality single-cell suspensions, utilization of comprehensive staining panels, and optimization of flow cytometry voltage. We then detail procedures for compensation adjustments and the implementation of effective gating strategies.

Before you begin

The protocol below describes the specific steps for preparing single cell suspensions of conjunctiva and lacrimal gland, and conducting flow cytometry analysis using wild type C57BL/6 mice. Additionally, we have successfully applied this protocol in dry eye mouse model.

Institutional permissions

Mice were fed in the specific pathogen-free animal room. The experimental protocols were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and were approved by Peking University Third Clinical Medical School Ethical Committee of Animals.

Mice

We typically employ female mice in our dry eye research due to their perceived increased vulnerability compared with male mice, as widely acknowledged in this field. A usual sample includes two pieces of conjunctiva or lacrimal gland. Initially, one may begin by harvesting samples from 4 to 6 mice at a time and gradually increase the number to 12 to 14 per session when proficiency is gained in all procedures.

Preparation on the prior day of mice euthanasia

Timing: 1 h

Researchers have to ensure that all required instrument and antibodies will be ready and available in enough quantities for the planed experiment.

• 1.Begin by preparing dissecting instruments, which should include ophthalmic scissors, toothed ophthalmic forceps, micro scissors, toothed micro forceps, and regular scissors. Subsequently, sterilize these instruments using a high-pressure steam sterilizer (Figure 1).
• 2.Gather the flow cytometry antibodies required for the experiments, covering main leukocyte subsets, myeloid cells, effector T cells, regulatory T cells, and Innate lymphoid cells (See Tables 1, 2, 3, 4, and 5 to get the list of antibodies for different panels). Ensure an adequate supply of antibodies corresponding to the number of samples.
• 3.Dissolve Collagenase D and DNase I in RPMI medium, achieving a concentration of 100 mg/mL. Perform this step in a clean bench, dispense the reconstituted solutions into 200 μL microcentrifuge tubes, and store them at −20°C for up to 3 months. Avoid repeated freezing and thawing since activity decreases after reconstitution.

Figure 1.

Dissecting instruments

Ophthalmic scissors, toothed ophthalmic forceps, two micro scissors, toothed micro forceps, and regular scissors are included.

Table 1.

Main leukocyte subsets panel

Fluorophore	Marker	Clone	Dilution
APC/Cy7	FVD	N/A	1:1000
AF700	CD45	30-F11	1:500
FITC	CD3	17A2	1:200
BV510	CD19	6D5	1:200
PE/Cy7	CD11b	M1/70	1:200
PerCp/Cy5.5	CD11c	N418	1:200
PE	MHC II	M5/114.15.2	1:200
APC	FcεRIα	MAR-1	1:200
BV605	c-kit	ACK2	1:200
BV421	Siglec-F	S17007L	1:200

Table 2.

Myeloid cells panel

Fluorophore	Marker	Clone	Dilution
APC/Cy7	FVD	N/A	1:1000
AF700	CD45	30-F11	1:500
PE/Cy7	CD11b	M1/70	1:200
APC	CD11c	N418	1:200
PE	F4/80	BM8	1:200
PerCP/Cy5.5	Ly6G	1A8	1:200
FITC	Ly6C	HK1.4	1:200
BV510	CD8a	53–6.7	1:200
BV421	MHC II	M5/114.15.2	1:200

Table 3.

Effector T cells panel

Fluorophore	Marker	Clone	Dilution
APC/Cy7	FVD	N/A	1:1000
AF700	CD45	30-F11	1:500
FITC	CD3	17A2	1:200
PerCP	CD4	RM4-5	1:200
BV510	CD8a	53–6.7	1:200
BV421	TCR γ/δ	GL3	1:200
PE/Cy7	IFN-γ	XMG1.2	1:200
BV605	IL-4	11B11	1:200
APC	IL-17A	TC11-18H10.1	1:200
PE	IL-22	Poly5164	1:200

Table 4.

Regulatory T cells panel

Fluorophore	Marker	Clone	Dilution
APC/Cy7	FVD	N/A	1:1000
AF700	CD45	30-F11	1:500
FITC	CD3	17A2	1:200
PerCP	CD4	RM4-5	1:200
BV510	CD8	53–6.7	1:200
APC	CD25	PC61 5.3	1:200
PE	Foxp3	MF-14	1:200
PE-cy7	IL-10	JES5-16E3	1:200

Table 5.

Innate lymphoid cells panel

Fluorophore	Marker	Clone	Dilution
APC/Cy7	FVD	N/A	1:1000
AF700	CD45	30-F11	1:500
BV605	NK1.1	PK136	1:200
BV510	CD127	A7R34	1:200
PerCP/Cy5.5	GATA3	16E10A23	1:200
PE	RORγt	Q31-378	1:200
PE/Cy7	IFN-γ	XMG1.2	1:200
APC	IL-17A	TC11-18H10.1	1:200
BV421	IL-5	TRFK5	1:200
FITC	Lineage	145-2C11; RB6-8C5; RA3-6B2; Ter-119; M1/70	1:20

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
AF700 anti-mouse CD45 (1:500)	BioLegend	Clone: 30-F11; Cat: 103127; RRID: AB_493714
FITC anti-mouse CD3 (1:200)	BioLegend	Clone: 17A2; Cat: 100204; RRID: AB_312661
BV510 anti-mouse CD19 (1:200)	BioLegend	Clone: 6D5; Cat: 115545; RRID: AB_2562136
PE/Cy7 anti-mouse CD11b (1:200)	BioLegend	Clone: M1/70; Cat: 101215; RRID: AB_312798
PerCP/Cy5.5 anti-mouse CD11c (1:200)	BioLegend	Clone: N418; Cat: 117327; RRID: AB_2129642
APC anti-mouse CD11c (1:200)	BioLegend	Clone: N418; Cat: 117309; RRID: AB_313778
PE anti-mouse MHC II (I-A/I-E) (1:200)	BioLegend	Clone: M5/114.15.2; Cat: 107607; AB_313322
BV421 anti-mouse MHC II (I-A/I-E) (1:200)	BioLegend	Clone: M5/114.15.2; Cat: 107631; RRID: AB_10900075
APC anti-mouse FcεRIα (1:200)	BioLegend	Clone: MAR-1; Cat: 134315; RRID: AB_10640726
BV605 anti-mouse CD117 (c-kit) (1:200)	BioLegend	Clone: ACK2; Cat: 135121; RRID: AB_2562040
BV421 anti-mouse CD170 (Siglec-F) (1:200)	BioLegend	Clone: S17007L; Cat: 155509; RRID: AB_2810421
PerCP anti-mouse CD4 (1:200)	BD Pharmingen	Clone: RM4-5; Cat: 553052; RRID: AB_394587
BV510 anti-mouse CD8a (1:200)	BioLegend	Clone: 53-6.7; Cat: 100751; RRID: AB_2561389
BV421 anti-mouse TCR γ/δ (1:200)	BioLegend	Clone: GL3; Cat: 118120; RRID: AB_2562566
PE/Cy7 anti-mouse IFN-γ (1:200)	BioLegend	Clone: XMG1.2; Cat: 505825; RRID: AB_1595591
BV605 anti-mouse IL-4 (1:200)	BioLegend	Clone: 11B11; Cat: 504125; RRID: AB_2562101
APC anti-mouse IL-17A (1:200)	BioLegend	Clone: TC11-18H10.1; Cat: 506916; RRID: AB_536018
PE anti-mouse IL-22 (1:200)	BioLegend	Clone: Poly5164; Cat: 516404; RRID: AB_2124255
PE anti-mouse F4/80 (1:200)	BioLegend	Clone: BM8; Cat: 123109; RRID: AB_893498
PerCP/Cy5.5 anti-mouse Ly6G (1:200)	BioLegend	Clone: 1A8; Cat: 127615; RRID: AB_1877272
FITC anti-mouse Ly6C (1:200)	BioLegend	Clone: HK1.4; Cat: 128005; RRID: AB_1186134
FITC anti-mouse lineage cocktail (CD3e, Gr-1, CD11b, CD45R, TER-119) (1:20)	BioLegend	Clone: 145-2C11, RB6-8C5, M1/70, RA3-6B2, Ter-119; Cat: 133302; RRID: AB_10697030
FITC Armenian hamster IgG/ FITC Rat IgG2b/ FITC Rat IgG2a isotype controls for lineage (1:20)	BioLegend	Cat: 133302
BV605 anti-mouse NK1.1 (1:200)	BioLegend	Clone: PK136; Cat: 108739; RRID: AB_2562273
BV510 anti-mouse CD127 (IL-7Rα) (1:200)	BioLegend	Clone: A7R34; Cat: 135033; RRID: AB_2564576
PerCP/Cy5.5 anti-mouse GATA3 (1:200)	BioLegend	Clone: 16E10A23; Cat: 653811; RRID: AB_2563218
PE anti-mouse RORγt (1:200)	BD Pharmingen	Clone: Q31-378; Cat: 562607; RRID: AB_11153137
BV421 anti-mouse IL-5 (1:200)	BioLegend	Clone: TRFK5; Cat: 504311; RRID: AB_2563161
APC anti-mouse CD25 (1:200)	eBioscience	Clone: PC61 5.3; Cat: RM6005; RRID: AB_10373675
PE anti-mouse Foxp3 (1:200)	BioLegend	Clone: MF-14; Cat: 126403; RRID: AB_1089118
PE/Cy7 anti-mouse IL-10 (1:200)	BioLegend	Clone: JES5-16E3; Cat: 505025; RRID: AB_11149682
Rat anti-mouse CD16/32 (FcR block) (1:50)	BioLegend	Clone: 93; Cat: 101320; RRID: AB_1574975
eFluor 780 (APC/Cy7) fixable viability dye (FVD)	eBioscience	Cat: 65-0865-18
Chemicals, peptides, and recombinant proteins
Collagenase D	Roche	Cat: 11088866001
DNase I	Roche	Cat: 10104159001
Cell activation cocktail without brefeldin A (phorbol 12-myristate 13-acetate [PMA] and ionomycin) (500×)	BioLegend	Cat: 423302
Protein transport inhibitor (containing brefeldin A) (1000×)	BD Pharmingen	Cat: 555029
Percoll	Cytiva	Cat: 17089101
RPMI 1640 medium	Cytiva	Cat: SH30027.01
1 M HEPES buffer (pH7.2- 7.4)	Solarbio	Cat: H1095
100 X Penicillin-Streptomycin	Solarbio	Cat: P1400
Fetal bovine serum (FBS)	Gibco	Cat: 10099141C
10 X HBSS (with Ca2+ and Mg2+)	Gibco	Cat: 14065056
1 X PBS	Solarbio	Cat: P1020
Critical commercial assays
eFluor 780 fixable viability dye	eBioscience	Cat: 65-0865-18
Foxp3/transcription factor staining buffer set	eBioscience	Cat: 00-5523-00
IC fixation buffer and permeabilization buffer set	eBioscience	Cat: 88-8824-00
Experimental models: Organisms/strains
Mouse: C57BL/6N (female), 6–8 weeks old	Vital River Laboratory	Strain No: 213
Software and algorithms
FlowJo version v10.8.1	Tree Star	https://www.flowjo.com
Other
200 μL microcentrifuge tubes	Axygen	Cat:PCR-02-C
1.5 mL centrifuge tubes	Axygen	Cat:MCT-150-C
15 mL centrifuge tubes	NEST	Cat: 601052
50 mL centrifuge tubes	NEST	Cat: 602052
10 mL pipets	NEST	Cat: 327003
6-well plates	NEST	Cat: 701001
24-well plates	NEST	Cat: 702001
5 mL round bottom polystyrene tubes	BD Falcon	Cat: 352054
Ophthalmic scissors	66 Vision Tech	Cat: 54020
Toothed ophthalmic forceps	66 Vision Tech	Cat: 53076
Micro scissors	66 Vision Tech	Cat: 54140B
Toothed micro forceps	66 Vision Tech	Cat: 53329A
Regular scissors	N/A	N/A
Insulin needle (30 gauge)	BD	Cat: 328431
Hamilton microsyringe (900 series)	Hamilton	Cat: 80360
1 mL manual pipette	Eppendorf	Cat: 3123000063
200 μL manual pipette	Eppendorf	Cat: 3123000055
20 μL manual pipette	Eppendorf	Cat: 3123000098
10 μL manual pipette	Eppendorf	Cat: 3123000020
2.5 μL manual pipette	Eppendorf	Cat: 3123000012
1 mL pipette tips	Axygen	Cat: T-1000-B
200 μL pipette tips	Axygen	Cat: T-200-Y
20 μL pipette tips	Axygen	Cat: T-400-R-S
200-mesh nylon filter (about 70 μm)	EASYBIO	Cat: BE6137-200
300-mesh nylon filter (about 48 μm)	EASY BIO	Cat: BE6137-300
Miltenyi C tubes	Miltenyi	Cat: 130-093-237
gentleMACS dissociator	Miltenyi	N/A
Stereo microscope	Leica Microsystems	N/A
Sterile clean bench	Haier Biomedical	N/A
Cell culture incubator	Thermo Scientific	N/A
Ice maker machine	PHCbi	N/A
High-pressure steam sterilizer	Hirayama	N/A
-20/4°C refrigerator	Haier Biomedical	N/A
Thermo Scientific Heraeus Multifuge X1R centrifuge	Thermo Scientific	N/A
Eppendorf 5424R centrifuge	Eppendorf	N/A
BD FACSCanto plus	BD Biosciences	N/A

Alternatives: We use most of antibodies from BioLegend but other providers are also available. We use Centrifuge tubes (15 mL and 50 mL) and culture plates from NEST, but alternatives from Corning and Costar are also available. 70 μm Cell Strainer (Falcon, Cat: 352350) and 40 μm Cell Strainer (Falcon, Cat: 352340) are alternatives of 200- and 300- mesh nylon filters. Alternatives of scissor and forceps, pipette and pipette tips from other instrument providers are also available.

Materials and equipment

Flow cytometer

A 10 color (4-Blue, 3-Red, and 3-Violet) BDFACSCanto plus flow cytometer was used to acquire the fluorochrome stained cell samples. The configuration of the flow cytometer is as follows: 4 channels for Blue 488 nm laser (FITC, PerCP/Cy5.5, PE, and PE/Cy7), 3 channels for the Red 640 nm laser (APC, APC/Cy7, and Alexa Fluor 700), 3 channels for the Violet 405 nm laser (Brilliant Violet 605, Brilliant Violet 421, Brilliant Violet 510).

Digestion solution

Reagent	Final concentration	Amount
RPMI medium	N/A	23.125 mL
HEPES (1 M)	10 mM	0.25 mL
Penicillin-Streptomycin (100×)	1×	0.25 mL
Inactivated FBS	3%	0.75 mL
Collagenase D (100 mg/mL)	2 mg/mL	0.5 mL
DNase I (100 mg/mL)	0.5 mg/mL	0.125 mL
Total	N/A	25 mL

Note: Prepare fresh and store at 4°C for up to 1 hour. FBS in all procedures should be heat inactivated at 56°C for 30 minutes to eliminate complement activity. The volume of solution is appropriate for 12 samples obtained from the conjunctiva and lacrimal gland of 6 mice.

RPMI enrich medium

Reagent	Final concentration	Amount
RPMI medium	N/A	5.70 mL
HEPES (1 M)	10 mM	0.06 mL
Penicillin-Streptomycin (100×)	1×	0.06 mL
Inactivated FBS	3%	0.18 mL
Total	N/A	6 mL

Note: Prepare fresh and store at 4°C for up to 1 day. FBS in all procedures should be heat inactivated at 56°C for 30 minutes to eliminate complement activity. The volume of solution is appropriate for 6 samples collected from lacrimal gland of 6 mice.

RPMI full culture medium

Reagent	Final concentration	Amount
RPMI medium	N/A	44.5 mL
Penicillin-Streptomycin (100×)	1×	0.5 mL
Inactivated FBS	10%	5 mL
Total	N/A	50 mL

Note: Prepare in a sterile clean bench and store at 4°C for up to 1 month.

RPMI activation medium

Reagent	Final concentration	Amount
RPMI full culture medium	N/A	6 mL
Cell Activation Cocktail without Brefeldin A (phorbol 12-myristate 13-acetate (PMA) and ionomycin) (1000×)	1×	12 μL
Protein Transport Inhibitor (Containing Brefeldin A) (500×)	1×	6 μL
Total	N/A	6 mL

Note: Prepare fresh in a sterile clean bench and store at 4°C for up to 1 day. This solution is used when the cells need to be cultured for 4 hours to detect cytokines in cytoplasm (Tables 3, 4, and 5). The volume of medium is suitable for 12 samples.

100% Percoll preparation solution

Reagent	Final concentration	Amount
Percoll	N/A	8.8 mL
HEPES (1 M)	10 mM	0.1 mL
Penicillin-Streptomycin (100×)	1×	0.1 mL
HBSS (10×)	1×	1 mL
Total	N/A	10 mL

Note: Prepare fresh each time and store it at 4°C for up to 1 day.

80% Percoll working solution: add 2.5 mL PBS (1×) to 10 mL 100% Percoll preparation solution.

Note: Prepare fresh each time and store it at 20°C–24°C for up to 6 hours. The volume of 80% Percoll working solution is suitable for processing 6 samples from lacrimal gland of 6 mice.

FACS buffer

Reagent	Final concentration	Amount
PBS (1×)	N/A	147 mL
inactivated FBS	2%	3 mL
Total	N/A	150 mL

Note: Prepare fresh in a sterile clean bench and store it at 4°C for up to 2 days.

IC fixation solution: This solution is utilized for staining with antibodies targeting intracellular cytokines, including IFN-γ, IL-4, IL-17A, IL-22.

1 X Foxp3 fixation/permeabilization working solution: combine 1 part of Foxp3 Permeabilization Concentrate (4×) with 3 parts of Foxp3 Fixation/Permeabilization Diluent.

Note: Prepare fresh each time and store it at 20°C–24°C up to 1 day. This solution is employed for staining with antibodies targeting transcription factors and nuclear proteins, including Foxp3, RORγt, and GATA3. Additionally, antibodies against intracellular cytokines can also be applied after using this solution.

1 X permeabilization working solution: combine 1 part of permeabilization buffer (10×) with 9 parts of double distilled water.

Note: Prepare fresh and store it at 20°C–24°C up to 1 day. This solution is used for staining with antibodies against intracellular cytokines or transcription factors following appropriate fixation.

Step-by-step method details

Conjunctiva and lacrimal gland dissection

Timing: 10 min/mouse

This procedure outlines the procedure for efficiently and thoroughly obtaining the conjunctiva and lacrimal gland.

• 1.Euthanize the mouse by cervical dislocation.
• 2.Under the Stereo microscope, inject 20 μL of PBS beneath the conjunctiva using an insulin needle or Hamilton microsyringe, either through the upper or lower eyelid conjunctival fornix (Figure 2A).

Note: This step separates the conjunctiva from the subconjunctival tissue, thereby reducing bleeding during dissection.

• 3.
  Dissection of the conjunctiva (Methods video S1).

  • a.
    Adding one drop of PBS on the surface of the conjunctiva (Figure 2B-left).

    Note: This step helps to amplify the field of view.

  • b.
    Grip the lacrimal sac in the inner canthus using microsurgical toothed micro forceps with one hand.
  • c.
    Employ a micro scissor to create a small incision (Figure 2B-right).
  • d.
    Cut along the junction of the conjunctiva and cornea, as well as along the lower margin of the tarsal plate (Figure 2C).
  • e.
    After cutting toward the outer canthus, alter the mouse’s orientation from inner to outer canthus.
  • f.
    Continue cutting toward the inner canthus (Figure 2D).
  • g.
    Completely excise the conjunctiva and place it in a 1.5 mL centrifuge tube with ice-cold PBS for preservation on ice (Figure 2E).
• 4.
  Dissection of the lacrimal gland (Methods video S2).

  • a.
    Elevate the skin using toothed ophthalmic forceps approximately 5 mm below the midpoint of the line connecting the outer canthus to the anterior edge of the auricle (Figure 2F).
  • b.
    Utilize ophthalmic scissors to excise a 5 mm × 5 mm section of skin.
  • c.
    Locate the lacrimal gland directly, and identify the transparent lacrimal duct connected to the outer canthus to confirm the presence of lacrimal gland tissue (Figure 2G).
  • d.
    Use a micro scissor to sever the connection between the lacrimal gland and the duct.
  • e.
    Gently grasp and extract the lacrimal gland with toothed micro forceps.
  • f.
    Place the lacrimal gland in a 1.5 mL centrifuge tube with ice-cold PBS for preservation on ice (Figure 2H).

CRITICAL: Bleeding may occur during the dissection of the conjunctiva owing to its rich blood supply. Gently wiping with a tissue can be helpful, but there is no necessity to wait until the bleeding completely ceases. For subsequent analysis, such as myeloid cells or T subsets, one sample pooled with two pieces of conjunctiva or lacrimal gland is adequate. If the target cells are relatively scarce, pooling more tissues in one sample is anticipated.

Figure 2.

Dissection of the conjunctiva and lacrimal gland

(A) Lifting the conjunctival fornix after injecting PBS beneath the conjunctiva.

(B) Making a small incision in the lacrimal sac at the inner canthus.

(C) Cutting along the limbus and the lower margin of the tarsal plate to obtain the majority of the conjunctiva.

(D) After cutting to the outer canthus, rotating the mouse and continuing cutting toward the inner canthus.

(E) Two pieces of conjunctiva from a mouse.

(F) Locating the skin of the lacrimal gland.

(G) Finding the lacrimal gland directly after excising the skin (white dashed circle), with the transparent lacrimal duct noticed beside the blood vessel (black arrow).

(H) Two pieces of lacrimal gland from a mouse.

Preparation of single cell suspensions using a Gentle MACS dissociator

Timing: 1.5 h/12 samples

This step describes the process for preparing high-quality single cell suspensions of conjunctiva and lacrimal gland.

• 5.Use micro scissors to cut the samples in a 1.5 mL centrifuge tube on ice (Figure 3A).

Note: Ensure that samples appear fragmented after cutting. 1.5 minutes per sample is usually appropriate.

• 6.Centrifuge at 4°C, 3500 rpm (1150 g), for 5 min (Eppendorf 5424R centrifuge).

Note: During centrifugation, prepare 25 mL Digestion solution for tissue dissociation of 12 samples.

• 7.Place samples on ice without shaking and position them beside of Gentle MACS dissociator.
• 8.Turn on the dissociator.
• 9.Aspirate the supernatants and discard (Figure 3B).
• 10.Add 1 mL Digestion solution using a tip-cut pipette tip by a regular scissor (Figure 3C) to the 1.5 mL centrifuge tube.
• 11.Resuspend the sample and transfer it to a Miltenyi C tube.
• 12.Aspirate 1 mL of Digestion solution again. Wash the residual tissue in the 1.5 mL centrifuge tube.
• 13.Transfer it to the C tube so that there is a total of 2 mL Digestion solution in each C tube (Methods video S3; Figure 3D).
• 14.Tighten the cap of the C tube.
• 15.Horizontally shake the C-tube to evenly mix the tissue.
• 16.Invert the C tube up and down.
• 17.Insert the C tube into the trough of the Gentle MACS dissociator (Methods video S4).
• 18.Set the heater around the C tube in the trough.
• 19.Select the program suitable for murine conjunctiva or lacrimal gland: 37_Multi_A_01 (41 min 10 s, 1332 rpr), and initiate sample dissociation under 37°C (Figure 3E). Refer to the manual for further details.
• 20.Once the program concludes, remove the heater, and then remove the C tube from the trough and place it on the centrifuge tube track (Methods video S5).

Note: Ensure no tissue pieces remain, and the solution appears as a cloudy cell suspension.

• 21.Add 5 mL FACS buffer into the C tube to dilute and neutralize the Digestion solution.
• 22.Gently invert upside down to ensure thorough cell suspension (Methods video S6).

Note: Avoid vortexing to minimize foam formation.

• 23.Apply the sample suspension to a 200-mesh nylon filter positioned on a 15 mL centrifuge tube to eliminate cellular debris (Methods video S7; Figures 3F and 3G).
• 24.Discard the nylon filter and centrifuge cell suspension at 4°C, 1700 rpm (543 g), for 5 min (Thermo Scientific Heraeus Multifuge X1R Centrifuge) (Figure 3H).
• 25.Completely aspirate the supernatants and discard.
• 26.Wash the cells once with 4 mL cold FACS buffer and spin down at 4°C, 1700 rpm (543 g), for 5 min.
• 27.Aspirate the supernatant and resuspend conjunctival cells in 1 mL FACS buffer and place samples on ice (Figure 3I).

Note: About 200,000 to 30,000 live cells including plenty of epithelial cells are expected for two pieces of conjunctiva at this time point, whether in control mice or in dry eye mice.

• 28.
  Enrich immune cells in lacrimal gland by Percoll due to its abundant impurities.

  • a.
    Resuspend lacrimal gland cells in 1 mL RPMI enrich medium and add 1 mL of 80% Percoll working solution (for a final Percoll concentration of 40%).
  • b.
    Mix thoroughly by pipetting and transfer the mixture into a new 5 mL round bottom polystyrene tube.
  • c.
    Carefully and slowly underlay with another 1 mL of 80% Percoll working solution. A clear separation of layers should be observed (Methods video S8; Figure 4A).
  • d.
    Carefully place the 5 mL round bottom polystyrene tube into the adapters of benchtop centrifuge using toothed ophthalmic forceps (Figure 4B). Avoid significant shaking.
  • e.
    Select the acceleration level of 9 and deceleration level to 0 (no brake, this is essential).
  • f.
    Centrifuge at 20°C–24°C, 2500 rpm (1174 g), for 20 min. The total centrifugation time is approximately 40 min (Figure 4C).
  • g.
    Carefully transfer the 5 mL round bottom polystyrene tube from the adapters to the centrifuge tube track. Immune cells will appear as an opaque ring between the 40% and 80% Percoll fraction (Figure 4D).
  • h.
    Carefully collect the immune cells at the interphase using 1 mL pipet and transfer to a fresh centrifuge 15 mL tube (Methods video S9).
  • i.
    Wash the cells once with 4 mL of cold FACS buffer and spin down at 4°C, 1700 rpm (543 g), for 5 min.
  • j.
    Resuspend the cells in 1 mL FACS buffer and place it on ice.

Note: Count the cell with a hemocytometer if necessary. After enrichment, about 80,000 to 100,000 live cells are expected for two pieces of lacrimal gland at this time point, whether in control mice or in dry eye mice.

Optional: In the absence of a Gentle MACS dissociator, manual dissociation is recommended. Transfer the tissues into a 6-wells plate containing 1 mL of Digestion solution (Step 11), and add an additional 1 mL Digestion solution. Shake the plate for 1 hour on a platform shaker at 200 rpm, 37°C. After digestion, very few tissue pieces should remain, and the solution should appear as a cloudy cell suspension. Transfer the contents to a fresh 15 mL centrifuge tube, shake vigorously for 1 minutes using a vortex, and apply the sample onto a 200-mesh nylon filter (Step 23). However, we have hardly experience on the comparative number of cells by this alternative procedure.

CRITICAL: Ensure the C tube cap is tightly secured to prevent solution leakage, which may lead to failed dissociation. The duration of dissociation is crucial, as prolonged dissociation exceeding 2 hours can result in severe cell death. Steps 20 to 23 should not exceed 10 minutes to maintain the maximum viability of cells.

Figure 3.

Preparation of single cell suspensions

(A) Samples should appear fragmented after thorough cutting.

(B) Aspirating the supernatants while avoiding aspirating the precipitation.

(C) Using a tip-cut pipette tip to transfer samples to a Miltenyi C tube, preventing samples from plugging at the tip of the pipette.

(D) Samples in Digestion solution in the C tube before dissociating.

(E) The selected program: 37_Multi_A_01 (41 min 10 s, 1332 rpr).

(F) The sterilized 200-mesh nylon filter.

(G) Straining the cells to remove cellular debris.

(H) Conjunctival cells after centrifugation.

(I) Conjunctival cells in 1 mL FACS buffer, ready for stimulation or staining.

Figure 4.

Enrich immune cells in lacrimal gland by Percoll

(A) A clear separation of layers should be observed before centrifugation.

(B) The sample tube in the adapters of the benchtop centrifuge.

(C) Setting the deceleration level to 0.

(D) Immune cells appearing as an opaque ring between the 40% and 80% Percoll fraction after centrifugation.

Stimulation and activation of immune cells

Timing: 4–6 h

This procedure is required for intracellular cytokine analysis. If not needed, proceed to the flow cytometry multi-color staining.

• 29.Centrifuge cells in 1.5 mL centrifuge tubes at 4°C, 3500 rpm (1150 g), for 5 min.
• 30.Aspirate the supernatants and discard.
• 31.Resuspend cells in 0.5 mL RPMI activation medium and transfer to a 24-well plate, place the plate in a 37°C incubator for 4–6 h of stimulation.
• 32.Transfer cells into 1.5 mL centrifuge tube and place on ice.

Flow cytometry multi-color staining

Timing: 3.5 h/12 samples

This procedure describes how to perform FACS staining of freshly prepared conjunctiva and lacrimal gland for flow cytometry using Fixable Viability Dye and fluorescence-conjugated antibodies.

• 33.Centrifuge at 4°C, 3500 rpm (1150 g), for 5 min and discard supernatants.
• 34.Resuspend the cells in 100 μL FACS buffer containing 2 μL anti-mouse CD16/CD32 for 10 min on ice, to block non-specific binding of immunoglobulin to the Fc receptors.
• 35.Idem and for the following spin.
• 36.Stain the cells in 50 μL FACS buffer containing Fixable Viability Dye and fluorescence-conjugated antibodies against cell-membrane molecules.
• 37.Incubate the cells for 20 min at 4°C in the dark, vortex every 10 min.
• 38.Wash the cells with 500 μL FACS buffer. Centrifuge at 4°C, 3500 rpm (1150 g), for 5 min and discard supernatants.
• 39.For intracellular cytokines staining only, resuspend the cells in 100 μL IC Fixation buffer. For transcription factors staining with or without intracellular cytokines staining, resuspend the cells in 100 μL 1 X Foxp3 fixation/permeabilization working solution.
• 40.Fixation lasts for 30 min at 20°C–24°C in the dark, vortex every 15 min.
• 41.Wash the cells with 300 μL 1 X permeabilization working solution. Centrifuge at 4°C, 7000 rpm (4602 g), for 5 min and discard supernatants.
• 42.Repeat Step 41.
• 43.Stain the cells in 50 μL 1 X permeabilization working solution containing fluorescence-conjugated antibodies against intracellular cytokines or transcription factors.
• 44.Incubate the cells for 40 min at 20°C–24°C in the dark, vortex every 20 min.
• 45.Repeat Step 41.
• 46.Resuspend the cells in 200 μL 1 X PBS, strain the cells to fresh 1.5 mL centrifuge tubes through a 300-mesh nylon filter. Keep cells at 4°C until data collection.

Optional: Staining in U-bottom 96-well plate is also feasible and can be more convenient with a multi-channel pipette. However, staining in 1.5 mL centrifuge tubes can minimize cell loss, especially after fixation and permeabilization.

Data collection on flow cytometer

Timing: 1–2 h/12 samples

This procedure describes the details in acquiring FACS data on Flow cytometer.

• 47.Acquire immune cells from the conjunctiva or lacrimal gland on BD FACSCanto plus.
• 48.Set the appropriate PMT voltage using single stained controls.

CRITICAL: Forward Scatter (FSC) and Side Scatter (SSC) voltage can change from one cytometer to another. Generally, the voltage of FSC should be relatively small, in order to gate the cells in the bottom left (Figures 5A and 6A). Ensure optimal voltage for other parameters using single stained samples.

• 49.Build a compensation matrix using single stained controls.

Optional: The compensation beads can also be employed for setting compensation.

• 50.The total live immune cells (CD45+) from two pieces of conjunctiva or lacrimal gland is no more than 10,000. Therefore, acquire the maximum events from each sample and analyze data using FlowJo Version v10.8.1.

Note: Compensation matrix can be further corrected in FlowJo.

Alternatives: Data collected by Flow cytometer can also be analyzed using Kaluza (Beckman Counter).

Figure 5.

The gating strategy for conjunctival effector T cells

(A) Debris is initially gated out by SSC-A versus FSC-A.(B) Doublets are excluded through FSC-A versus FSC-W gating.(C) FVD negative cells are gated as live cells.(D) CD45+ cells are gated as immune cells.(E) CD3+ cells are identified as T cells.(F) CD8+CD4-, CD4+CD8-, and CD8-CD4- cells are gated as CD8+ T, CD4+ T, CD8-CD4- T cells.(G) CD8+IFN-γ+ cells are gated as Tc1 cells, CD8+IL-4+ cells (Tc2 cells) are not found.(H) CD8+IL-17+ cells (Tc17 cells) and CD8+IL-22+ cells (Tc22 cells) are not found.(I) CD8-CD4-TCR γδ- T cells produce IFN-γ but not IL-4.(J) CD8-CD4-TCR γδ+ T cells are gated as  γδ T cells.(K) CD4+IFN-γ+ cells are gated as Th1 cells, CD4+IL-4+ cells (Th2 cells) are not found.(L) CD4+IL-17A+ cells are gated as Th17 cells, CD4+IL-22A+ cells are gated as Th22 cells.(M) CD8-CD4-TCR γδ- T cells produce IL-17A but not IL-22.(N) TCR γδ+IFN-γ+ cells are gated as γδ T1 cells, TCR γδ+IL-4+ cells (γδ T2 cells) are not found.(O) TCR γδ+IL-17A+ cells are gated as γδ T17 cells, TCR γδ+IL-22A+ cells are gated as γδ T22 cells.Re-use of the supplementary figure 5 in the prior manuscript has obtained permission from the publisher.

Figure 6.

The gating strategy for conjunctival ILCs

(A) Debris is initially gated out by SSC-A versus FSC-A.(B) Doublets are excluded through FSC-A versus FSC-W gating.(C) FVD negative cells are gated as live cells.(D) CD45+ cells are gated as immune cells.(E) Lineage isotype antibodies are applied as negative control.(F)  Lineage (CD3e, Gr-1, CD11b, CD45R, TER-119) negative cells are gated as ILC cells.(G) Lineage-NK1.1+CD127- cells are gated as NK cells, Lineage-NK1.1+CD127+ (ILC1) are not found.(H) NK cells produce IFN-γ and IL-5.(I) Lineage-NK1.1-CD127-GATA3- cells are not found.(J) Lineage-NK1.1-CD127-IL-5+ cells are gated as ILC2, Lineage-NK1.1-CD127-RORγt+ cells are gated as ILC3.(K) A significant proportion of ILC3 cells produce IL-17A.Re-use of the supplementary figure 7 in the prior manuscript has obtained permission from the publisher.

Expected outcomes

Effector T cells panel: Debris is initially gated out by SSC-A versus FSC-A. An appropriate gate for SSC-A versus FSC-A is positioned at the bottom left, not only because the above PMT voltage is relatively low but also because cells tend to shrink after the fixation and permeabilization steps. The gate is relatively narrow due to the comparable sizes of different T cell subsets, unlike the significant variance observed among myeloid cells. Doublets are then excluded through FSC-A versus FSC-W gating. Subsequently, dead cells are excluded by staining with Fixable Viability Dye. CD45+ cells are gated as immune cells, and CD3+ cells are identified as T cells. Further plotting for CD8+, CD4+, and TCR γδ+ allows the distinction of CD8+ T, CD4+ T, γδ T, and other T (CD8-CD4-TCR γδ-, NKT as inferred) cell subsets. Under normal conditions, Tc1 (IFN-γ+) cells are more abundant than Tc2 (IL-4+), Tc17 (IL-17A+), or Tc22 (IL-22+) among CD8+ T cells in the conjunctiva and lacrimal gland. Effector CD4+ cells can be categorized into Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and Th22 cells (IL-22+) in this panel. γδ T17 (IL-17A+) are the predominant γδ T cells. CD8-CD4-TCR γδ- T cells also produce IFN-γ or IL-17A. The gating strategy for conjunctival effector T cells is illustrated in Figure 5. The frequencies of immune cell subsets can vary significantly due to different strains, feeding conditions, and even different SPF houses from different laboratories or companies. Mice in this protocol are bought from Vital River Laboratory and directly euthanized once they are shipped to our laboratory.

Innate lymphoid cells panel: Debris is initially gated out by SSC-A versus FSC-A, and the gating strategy for the ILCs panel is similar to that of the effector T cells panel. Doublets are then excluded through FSC-A versus FSC-W gating, and dead cells are subsequently excluded with Fixable Viability Dye staining. CD45+ cells are then gated as immune cells. ILC cells are further gated as Lineage (CD3e, Gr-1, CD11b, CD45R, TER-119) negative cells. Further plotting for NK1.1 and CD127 enables the distinction of NK cells (NK1.1+CD127-), ILC1 (NK1.1+CD127+), and NK1.1-CD127- cells. It is important to note that NK cells also belong to ILCs. ILC1 is rarely found in the conjunctiva and lacrimal gland. Among NK1.1-CD127- ILCs, ILC2 (IL-5+) are more prevalent than ILC3 (RORγt+), and a significant proportion of ILC3 cells produce IL-17A. The gating strategy for conjunctival ILCs is illustrated in Figure 6.

Examples of main leukocyte subsets panel, myeloid cells panel, and regulatory T cells panel can be found in supplementary materials in our previous study (https://www.tandfonline.com/doi/full/10.1080/09273948.2023.2182327). Re-use of all the supplementary figures in the prior manuscript has obtained permission from the publisher.

Note: The cell viability of conjunctiva varies from 20% to 30% in representative plots of Figures 5 and 6. We reanalyze the viability of CD45 positive immune cells in conjunctiva and lacrimal gland (Myeloid cells panel and Effector T cells panel) (Figure 7). Results show abundant impurities in conjunctiva lead to the lower viability compared with lacrimal gland, which is purified by Percoll (Figure 7A versus 7B; Figure 7C versus 7D). Meanwhile, stimulation and activation induce a higher percentage of dead cells both in conjunctiva and in lacrimal gland (Figure 7C versus 7A; Figure 7D versus 7B), due to activation-induced cell death (AICD) as inferred.

Figure 7.

The viability of CD45+ pan-immune cells for Myeloid cells panel and Effector T cells panel in conjunctiva and lacrimal gland

(A) The viability of CD45+ pan-immune cells for Myeloid cells panel in conjunctiva.

(B) The viability of CD45+ pan-immune cells for Myeloid cells panel in lacrimal gland.

(C) The viability of CD45+ pan-immune cells for Effector T cells panel in conjunctiva.

(D) The viability of CD45+ pan-immune cells for Effector T cells panel in lacrimal gland.

Quantification and statistical analysis

FlowJo Version v10.8.1 (LLC https://www.flowjo.com) was used for data analysis.

Limitations

Firstly, conjunctiva and lacrimal gland in this protocol are available for Flow cytometry, RNA extraction, and protein extraction. However, if the aim is to perform immunofluorescence staining, conjunctiva should be dissected completely together with the eyeball and eyelid. Secondly, inactivated FBS was included in the Digestion solution in prior experiments and this protocol. However, after receiving comments from reviewer that FBS may impair the protease activity of collagenase D and DNase I, we discussed with several immune laboratories in Peking University and Tsinghua University that Digestion solution without FBS tends to be more effective. Therefore, the experimenter could try conducting the experiment using Digestion solution without FBS. Removing FBS may increase cell recovery, but it may also increase cell mortality. Thirdly, the analysis primarily focuses on major subsets of immune cells in the conjunctiva and lacrimal gland, with a specific emphasis on T cells and ILCs. B cells, which can be further subdivided into B-1 and B-2 subsets, are not covered in this protocol. Additionally, Tfh cells are excluded as they are predominantly found in secondary lymphoid tissues like lymph nodes, where they stimulate B cells. For a more detailed exploration, full spectrum flow cytometry or Single-cell RNA sequencing can be employed. Furthermore, aside from the conventional classification system, Hu et al. (2023)² proposed alternative theories for immune cell subdivision. Fourthly, it is crucial to optimize antibody panels for the specific flow cytometer being used, as lasers and voltage settings may vary among different instruments. Utilizing single stained controls and fluorescence minus one (FMO) controls for all antibodies is highly recommended. Fifthly, while this flow cytometry protocol provides insights into resident immune cells throughout the entire tissue, incorporating immunofluorescence staining can aid in understanding the distribution of immune cells in different layers of the conjunctiva and lacrimal gland.

Troubleshooting

Problem 1

Bleeding complicates the dissection of the conjunctiva (step 3).

Potential solution

Bleeding is a common challenge during conjunctiva dissection, causing temporary visual obstruction. Though often unavoidable, placing a tissue beside the conjunctiva to absorb the bleeding can help maintain a clear vision for several seconds. Alternatively, an assistant can perform topical irrigation using saline with a 5 mL syringe, providing additional assistance in managing the issue.

Problem 2

Liquid droplets appear in the trough, and little liquid is left in the C tube (step 20).

Potential solution

Ensure a tight closure of the C tube beyond first resistance. The top area of the cap and the C tube must be clean for proper functioning of the seal. After inserting the C tube into the trough, it should be securely fixed through the snap-fit mechanism. While not directly associated with the repeated sterilization and reuse of C tubes, we recommend limiting the sterilization of C tubes to no more than five times.

Problem 3

No obvious opaque ring or separation of layers between the 40% and 80% Percoll fraction is observed (step 28g), but obvious cell precipitation can be observed at step 24 (Figure 3H).

Potential solution

It indicates that something is wrong during the enrichment of immune cells by Percoll (Step 28). Ensure the Percoll solution is freshly prepared. A clear separation of layers should be evident before centrifugation. Confirm the deceleration level is set to 0. In order to reverse this situation, transfer all 3 mL of liquids in the 5 mL round bottom polystyrene tube to 30 mL 1 X PBS in a new 50 mL centrifuge tube and centrifuge at 4°C, 1700 rpm (543 g), for 5 min. Observe for obvious precipitation. Then repeat step 28.

Problem 4

No positive staining of some markers by flow cytometry (step 48).

Potential solution

Proper voltage setting of the specific channel using single stained samples before data acquisition is essential. Ensure that antibodies are applied at the correct concentration. Pair low-expressing antigens with bright fluorochromes such as PE. Conduct pre-experiments to confirm that all targeted populations are detectable in fully stained samples.

Problem 5

The compensation matrix in the flow cytometer is not appropriate in FlowJo (step 50).

Potential solution

Check and correct the compensation matrix in FlowJo after data acquisition. Moreover, we recommend building the compensation matrix mainly in FlowJo rather than in the flow cytometer. The final gating and results are generated and exported in FlowJo. This means you can even disregard the compensation matrix and simply set a compensation value when acquiring data on flow cytometer. For detailed skills, adjust compensation among FVD channel and other channels first on the gate of single cells, then CD45 channel and other channels on the gate of single-live cells, and finally other channels on the gate of single-live immune cells.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hong Qi (doctorqihong@163.com).

Technical contact

Technical questions about this protocol should be directed to the technical contact, Baikai Ma (doctormabk@qq.com)

Materials availability

No new materials were generated in this protocol.

Data and code availability

The study did not generate analyze datasets or codes.