Materials and Reagents

20, 200, and 1,000 μl Pipette tips (no specific brand)
Thermo Scientific^TM screw cap microtubes (2.0 ml) (Fisher Scientific, catalog number: 21-403-202)
3 ml syringes (BD Biosciences, catalog number: 309657)
18 G 1.5 in needles (BD Biosciences, catalog number: 305196)
21 G 1.5 in needles (BD Biosciences, catalog number: 305167)
50 ml conical tubes (Crystalgen, catalog number: 23-2263)
12 × 75 mm (5 ml) round bottom polystyrene tubes (Corning, catalog number: 352052)
Petri dish (Falcon, catalog number: 351029)
Aluminum foil
Dispase powder (Gibco, catalog number: 17105041)
ChAT^BAC-eGFP mice (B6.Cg-Tg (RP23-268L19-EGFP) 2Mik/J) (The Jackson Laboratory, catalog number: 7902)
200 Proof Ethanol (Koptec, catalog number: V1001)
DNase I (Sigma, catalog number: 10104159001)
Dulbecco’s Phosphate-Buffered Saline (PBS) (Boston BioProducts, catalog number: BSS-220DM-C)
DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) (ThermoFisher Scientific, catalog number: 11320033)
Heat-inactivated fetal bovine serum (FBS) (ThermoFisher Scientific, catalog number: 10082139)
Tyrode’s Solution (with bicarbonate, HEPES, and 0.25% BSA, without calcium) (Boston BioProducts, catalog number: PY-912)
Papain from papaya latex (Sigma, catalog number: P3125)
L-cysteine (Sigma, catalog number: C7352)
Tyrode’s Solution (with HEPES and calcium) (Boston BioProducts, catalog number: BSS-355)
Leupeptin trifluoroacetate salt (leupeptin) (Sigma, catalog number: L2023)
HBSS, 1× without calcium, magnesium, and phenol red (Hank’s Balanced Salt Solution) (Corning, catalog number: 21-022-CV)
EDTA (0.5 M), pH 8.0. RNase-free (Thermo Fisher, catalog number: AM9260G)
TruStain FcX^TM (anti-mouse CD16/32) antibody (Biolegend, catalog number: 101320)
Pacific Blue anti-mouse CD45 monoclonal antibody (Biolegend, catalog number: 103126)
Allophycocyanin (APC) anti-mouse CD326 (EpCAM) monoclonal antibody (Biolegend, catalog number: 118214)
Propidium iodide (PI) (Sigma, catalog number: P4170)
70% EtOH (500 ml) (see Recipes)
Dispase solution (see Recipes)
DMEM-based stopping solution (see Recipes)
Tyrode buffer-based digesting solution with papain (see Recipes)
Tyrode buffer-based stopping solution (see Recipes)
FACS buffer (washing buffer for flow cytometry) (see Recipes)

Equipment

Dissection scissors (straight) (Fisher Scientific, catalog number: 08-951-5)
Forceps (straight, serrated) (Fisher Scientific, catalog number: 13-812-36)
Pipettes (P10, P200, P1000) (no specific brand)
Fisherbrand^TM Isotemp^TM General Purpose Deluxe Water Bath (Fisher Scientific, catalog number: FSGPD05)
Fisherbrand^TM Multiplatform Shaker (Fisher Scientific, catalog number: 88-861-021)
Dissecting microscope (Leica, catalog number: M165FC)
Scalpel (No.10) (ThermoFisher Scientific, catalog number: 3120032)
Fisher Vortex Genie 2 (Fisher Scientific, catalog number: 12-812)
100 μM Fisherbrand^TM Sterile Cell Strainers (Fisher Scientific, catalog number: 22-363-549)
Sorvall Legend X1R Centrifuge (ThermoFisher Scientific, catalog number: 75004261)
BD LSRFortessa^TM Flow Cytometer (BD Biosciences)

Software

FlowJo v.8 (FlowJo, LLC, https://www.flowjo.com)
Prism 7 (GraphPad Software, https://www.graphpad.com/scientific-software/prism/)

Procedure

Isolation of mouse snouts
Mice used: ChAT(BAC)-eGFP (B6.Cg-Tg(RP23- 268L19-EGFP)2Mik/J), 3-6 months of age of both sexes. All animal care and procedures are approved by the Animal Care and Use Committees (IACUC) of Brigham and Women’s Hospital.
1. Euthanize the mouse. Spray the fur over the abdomen with 70% EtOH (see Recipe 1). Make a midline abdominal incision with straight scissors to open the abdominal cavity and bleed the mice through puncture of the abdominal aorta. Allow the mouse to bleed for 1-2 min, for blood outflow from the head and avoidance/minimization of further necessity to lyse red blood cells.
2. Decapitate the mouse by cutting off the mandible and separating the head from the rest of the body.
3. Using straight scissors, cut off the tip of the nose and the front incisor teeth. Remove the skin of the head and release the skull from the surrounding muscle and brain tissues.
4. Cut off the zygomatic arches (Figure 1A). Remove the parietal bone. Make longitudinal incisions along the frontal bone, exposing the nasal cavity from the top (Figures 1A-1C).
  
  Figure 1. Representative images of nasal snout preparation for first digestion with dispase solution. A. The mouse head, released from the surrounding tissues and brain. The bilateral zygomatic arches need to be removed (black lines). The red dotted lines represent the longitudinal lines for further incision along the frontal bone. B. Longitudinal incision along the frontal bone. C. The frontal and nasal bones lifted, and the top of the nasal cavity exposed. D. The snout placed in 2 ml tube with pre-warmed dispase solution.
Nasal epithelial cell dissociation
1. Pre-warm 750 µl of dispase solution (see Recipe 2) in 2 ml tube at 37°C water bath for 5-10 min. Place the isolated snout into the solution (Figure 1D) and incubate on a shaker at 100 rpm for 30 min at room temperature. Keep the tubes protected from light with foil covers.
2. Reduce the digestion by adding 750 µl of cold DMEM-based stopping solution (see Recipe 3). Place the tubes on ice for 2-5 min.
3. Remove the digested snout out of the tube and place on Petri dish under a dissecting microscope with the palate facing down.
4. Remove the nasal bone comprising the superior wall of the nasal cavity. Now you can visualize the nasal cavity with the overlying mucosa (Figure 2A). Split the nasal cavity into left and right halves through a longitudinal incision along the palate (Figure 2B).
  
  Figure 2. Representative images of single cell preparations from isolated nasal mucosa of the snout. A. A scheme of the lateral wall of the nasal cavity, with red color indicating the nasal mucosa coverage. B. The nasal cavity split into right and left halves on a Petri dish. The mucosa was scraped off with scalpel under a dissecting microscope. The red outlines represent the area of the nasal mucosa to be isolated. C. Transfer of the scraped mucosa to a 2 ml tube. D. Flushing of the Petri dish with 750 µl of Tyrode buffer based digesting solution with papain. E. After stopping the digestion and vortexing the sample, trituration of the suspension with 18-gauge needle 8-10 times, followed by trituration with 21-gauge needles for 15-20 times. F. The cell suspension strained through 100 µm filter into a 50 ml conical tube.
5. Scrape the nasal mucosa with a scalpel until no tissue can be visualized under the dissecting microscope except for the bone. Transfer the separated mucosa with the small underlying bones and cartilages to a 2 ml tube (Figure 2C).
6. Flush the Petri dish with 750 µl of Tyrode buffer based digesting solution with papain (see Recipe 4) and transfer the flushed fluid into the tube containing the nasal mucosa (Figure 2D).
7. Incubate the isolated nasal mucosa in Tyrode based digestion buffer for 40 min at 37°C, shaking at 210 rpm.
8. Vortex the digested tissue at 300 rpm for 10 s. Terminate the digestion by adding cold Tyrode buffer-based stopping solution (see Recipe 5) and placing the sample on ice.
9. Vortex the tissue at 1,000 rpm for 20 s. Triturate the solution with a syringe attached to an 18-gauge needle 8-10 times. Remove the remaining bones and cartilages to avoid clogging the syringe. Switch the needle on the syringe to 21-gauge needle and triturate 15-20 more times (Figure 2E).
10. Pass the suspension through a 100 µm filter into a 50 ml conical tube (Figure 2F). Add 20-25 ml of cold FACS buffer (see Recipe 6).
11. Spin the tube at 350 × g for 15 min at 4°C. Check the tube for the visible pellet. Discard the supernatant without disturbing the cell pellet and resuspend the pellet in 1 ml of FACS buffer and transfer to a 5 ml polystyrene tube. Fill the polystyrene tube with 4 ml of FACS buffer.
12. Spin the tube at 350 × g for 10 min at 4°C. Discard the supernatant and resuspend the pellet in 100 µl of FACS buffer.
13. To block Fc receptors, pre-incubate the cell suspension with TruStain FcX^TM (anti-mouse CD16/32) antibody; use 1.0 µg of antibody per 10⁶ cells in 100 µl volume for 5-10 min on ice. Washing after this step is not necessary.
14. Incubate the cells with antibodies for 45 min on ice, protected from light. The panel of antibodies used for brush cell identification includes pacific blue anti-mouse CD45 monoclonal antibody at 0.25 µg per 10⁶ cells in 100 µl volume and allophycocyanin (APC) anti-mouse EpCAM monoclonal antibody at 0.5 µg per 10⁶ cells in 100 µl volume.
15. Wash the cells by resuspending them with 1 ml of FACS buffer and add an additional amount of 3.8-4 ml of cold FACS buffer. Spin at 350 × g for 10 min at 4°C. Discard the supernatant and resuspend the pellet in 300 µl of cold FACS buffer.
16. Perform manual cell counts if not planning on running the whole sample on FACS machine. If the whole sample is collected on the machine, the counting step can be omitted.
17. Add propidium iodide (PI) 5 µg/ml for viability for 2-3 min before FACS.

Data analysis

After isolation of the nasal mucosa and digestion with this two-step protocol (see the graphical abstract), label the cell suspension fluorescently with EpCAM and CD45. Cells are gated by FSC-A/SSC-A to exclude debris and by SSC-H/SSC-W and FSC-H/SSC-W to select single cells (Figure 3A). Propidium iodide (PI) is used as a dead cell exclusion marker. From 1.2 × 10⁶ to 3.1 × 10⁶ live cells can be obtained with this method (Figure 3B). In our analyses, CD45^low/negand EpCAM^high cells constituted 14-18% of all live cells. Brush cells were defined as EpCAM^highand GFP⁺cells, and comprised 14-24% of the EpCAM^highpopulation and 1.3% of all live cells. This method allows isolation of 16,000-30,000 GFP⁺ brush cells per mouse. Nasal brush cells can further be subdivided into SSC^low and SSC^high populations based on their size, and represent microvillous cells and solitary chemosensory cells, respectively. SSC^lowcomprised the majority of GFP⁺ cells (94%), while SSC^highcells represented 4% of brush cells.

Figure 3. Flow cytometry analysis of nasal brush cells. The cell suspensions were stained with fluorescent-conjugated antibodies against EpCAM and CD45 and examined by flow cytometry. The gating strategy was previously published (Ualiyeva et al., 2020). A. Representative flow cytometry gating strategy. Cells were gated based on their size and granularity on forward and side scatter features. Doublets were further excluded, and live cells were selected based on absence of staining with Propidium Iodide. CD45^low/neg were evaluated on their expression of EpCAM. Brush cells were defined as EpCAM^high GFP⁺ cells and were further specified on forward scatter and side scatter characteristics. SSC^high population represents solitary chemosensory cells, and SSC^low brush cells correspond to microvillous cells. B. Flow cytometry analysis of cell suspensions from C57BL6 mice. The sample was pre-gated on single live cells, and CD45^low/neg cells were evaluated on their expression of EpCAM. C. Number of live cells recovered from nasal mucosa and frequency of EpCAM^highepithelial cells as percent of live cells. D. Frequency of nasal brush cells presented as percent of live cells, percent of EpCAM^highpopulation and number of nasal brush cells per mouse nose. E. Frequency of SSC^high (solitary chemosensory cells) and SSC^low (microvillous cells) populations as percent of all EpCAM^high GFP⁺ cells and counts. Each dot represents a separate mouse. Data are from 3 separate experiments with 2 mice each.

This protocol has been successfully applied to isolate nasal brush cells for RNA sequencing and functional assays (Ualiyeva et al., 2020).

Recipes

70% EtOH (500 ml)
350 ml 200 proof pure ethanol
150 ml distilled H₂O
Mix well
This solution is flammable.
Dispase solution, prepare fresh prior to isolation
16 U/ml dispase powder
Dulbecco’s Phosphate-Buffered Saline (PBS)
Distilled H₂O
20 μg/ml DNase I
Prepare 1× PBS
Dissolve 16 U/ml dispase powder and 20 μg/ml DNase I in 1× PBS
Store solution at 4°C. Pre-warm in a water bath to 37°C before adding the snout.
DMEM-based stopping solution (DMEM with 5% FBS) (525 ml) (can be stored for up to 2 months at 4°C)
500 ml Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM)
25 ml Heat-inactivated fetal bovine serum (FBS)
Prepare solution in a sterile biosafety cabinet and store at 4°C
Tyrode buffer-based digesting solution with papain (prepare fresh prior to isolation)
Tyrode’s Solution (with bicarbonate, HEPES, and 0.25% BSA, without calcium)
20 μl/ml papain (28 U/mg)
10 μl/ml L-cysteine (25 mg/ml)
Prepare solution in a sterile biosafety cabinet and store at 4°C
Tyrode buffer-based stopping solution, prepare fresh prior to isolation
Tyrode’s Solution (with HEPES and calcium)
2 μl/ml leupeptin (5 mg/ml)
FACS buffer (washing buffer for flow cytometry), can be stored up to 1 month at 4°C
HBSS, 1× without calcium, magnesium, and phenol red (Hank’s Balanced Salt Solution)
2% Heat-inactivated fetal bovine serum (FBS)
2 mM Ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8.0)
Prepare solution in a sterile biosafety cabinet and store at 4°C

Acknowledgments

This protocol was adapted with minor modifications from a previous study published by Ualiyeva et al. (2020). This methodology was based on a previous protocol for tracheal brush cell isolation used in our lab (Ualiyeva et al., 2019). We thank Adam Chicoine at the Brigham and Women’s Human Immunology Center Flow Core for his help with flow cytometric sorting. This work was supported by National Institutes of Health Grants R01 HL120952 (N.A.B.), R01 AI134989 (N.A.B), U19 AI095219 (N.A.B., L.G.B), K08 AIl32723 (L.G.B), and T32 AI00730634 (S.U.), and by the American Academy of Allergy, Asthma, and Immunology (AAAAI)/American Lung Allergic Respiratory Disease Award (N.A.B.), by the AAAAI Foundation Faculty Development Award (L.G.B.), by the Steven and Judy Kaye Young Innovators Award (N.A.B.), by the Joycelyn C. Austen Fund for Career Development of Women Physician Scientists (L.G.B.), and by a generous donation by the Vinik family (L.G.B.).

Competing interests

The authors declare no competing interests.

Ethics

All procedures performed in animal study were guided by Brigham and Women’s Hospital IACUC protocol 2016N000517.

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