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Jan 2020

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Isolation of Nasal Brush Cells for Single-cell Preparations
用于单细胞制备的鼻刷细胞的分离    

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Abstract

Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays.


Graphic abstract:


Workflow of nasal digestion for brush cell isolation.


Keywords: Nasal brush cells (鼻刷细胞), Cholinergic epithelial cells (胆碱能上皮细胞), Microvillous cells (微绒毛细胞), Solitary chemosensory cells (孤立的化学感应细胞), Bitter taste sensing cells (苦味感知细胞), Bitter taste receptors (苦味受体), Tuft cells (Tuft细胞 )

Background

Brush cells are chemosensory epithelial cells best known for their expression of bitter taste receptors (Finger et al., 2003; Krasteva and Kummer, 2012). Brush cells are also be referred to as tuft cells and have been found in the airways (Krasteva and Kummer, 2012), gastrointestinal tract (Howitt et al., 2016), and urinary epithelium (Deckmann et al., 2014). Morphologically, brush cells are characterized by apical microvilli that form a tuft-like projection on the cell surface, extending to the mucosal lumen (Rhodin and Dalhamn, 1956; Reid et al., 2005). Protective functions of brush cells have been most closely linked to their generation of acetylcholine leading to activation of peptidergic sensory nerve fibers (Krasteva et al., 2011). We have previously reported a role for brush cell-derived IL-25 in eliciting type 2 inflammation and epithelial cell remodeling in the lung (Bankova et al., 2018). We recently reported robust generation of pro-inflammatory lipid mediators termed cysteinyl leukotrienes by nasal brush cells in response to aeroallergens and ATP through the purinergic receptor P2Y2 (Ualiyeva et al., 2020), suggesting potent functions for these cells in directing immune responses in the airways.


In the nose, the mucosal lining is composed of olfactory and respiratory epithelia (Adams, 1972). In mice, the olfactory epithelium lines up to 50% of the total nasal mucosa, overlying the middle and superior turbinates and the caudal/posterior area of the nasal septum, while the respiratory epithelium covers around 45% of the nasal cavity in more rostral/anterior parts (Gross et al., 1982; Chamanza and Wright, 2015). Solitary chemosensory cells in the respiratory mucosa of the nose respond to irritants and bacteria and are established as the nasal equivalent of tracheal brush cells in mice (Finger et al., 2003; Sbarbati and Osculati, 2003) and humans (Lee et al., 2014; Kohanski et al., 2018). Microvillous cells are related cells found in the apical olfactory epithelium, above the layer of olfactory sensory neurons, and supporting sustentacular cells (Hansen and Finger, 2008). Both microvillous cells and solitary chemosensory cells express Chat, choline acetyltransferase, an enzyme necessary for the synthesis of the neurotransmitter acetylcholine (Krasteva et al., 2011), Trpm5, a transient receptor potential gene ubiquitous in chemosensory cells (Pérez et al., 2002; Kaske et al., 2007; Zhang et al., 2007; Ogura et al., 2011; Genovese and Tizzano, 2018), and depend on Pou2f3, a transcription factor required for differentiation of all chemosensory cells in the nose, trachea, and intestine (Ohmoto et al., 2013; Yamaguchi et al., 2014; von Moltke et al., 2016).


Our recent study using fluorescence-activated cell sorting (FACS) based on the fluorescent reporter expression of Chat (ChAT-eGFP) and size and granularity characteristics [forward scatter (FSC) and side scatter (SSC)] allowed us to distinguish the olfactory-enriched microvillar cells from the respiratory-enriched solitary chemosensory cells. We confirmed by bulk RNA-sequencing (RNA-seq) that both populations belong to the larger family of chemosensory tuft/brush cells and are mostly distinguished by their varied expression of taste receptors. This protocol describes our method for isolation of a mixed population of nasal chemosensory brush cells providing high yields of ~20,000/mouse, which can be further characterized by FACS and RNA-seq for investigation of the similarities and differences between these cell types in the nasal cavity.


Several groups have reported successful isolation of solitary chemosensory cells from the respiratory nasal mucosa and microvillar cells from the olfactory mucosa. Gulbransen et al. (2008) used a Tyrode-based solution supplemented with 20 U/ml of papain to isolate solitary chemosensory cells from the respiratory mucosa of TRPM5-GFP fluorescent reporter mice. Several hundred viable cells were used for calcium flux studies in response to bitter tasting agonists. Lin et al. (2008) isolated solitary chemosensory cells from the anterior respiratory epithelium with a Ringer-based solution with 10-30 U/ml of papain to measure changes in intracellular calcium levels in single cells in response to odorous irritants. Ogura et al. (2011) isolated microvillar cells from the main olfactory epithelium of ChAT-eGFP and TRPM5-GFP mice with 4 U/ml of papain for 3-5 min at room temperature and showed increased calcium flux from freshly isolated cells in response to ATP, soil bacterium lysate, and denatonium benzoate. Although no information was provided for the total yields of recovered solitary chemosensory or microvillous cells with the described protocols, each used <100 cells for live imaging for calcium flux. We tested a comparable concentration of papain (26 U/ml) to isolate brush cells from the trachea and found that pre-incubation of the trachea with high dose dispase (16 U/ml) results in a 20-fold increase of isolated brush cell yields compared to digestion with papain alone (Ualiyeva et al., 2019).


Here, we describe a step-by-step protocol for isolation of brush cells from the murine snout of ChAT-eGFP fluorescent transgenic mice, with enhanced green fluorescent protein expression dictated by Chat. Choline acetyltransferase is highly expressed in both subsets of nasal chemosensory brush cells, in tracheal, intestinal, and urethral brush/tuft cells and specifically enriched in these chemosensory epithelial cells compared to other epithelial cells. This allows for identification of the fluorescent cholinergic brush cells by FACS. The single-cell solution preparation procedure is based on initial incubation of the snout with dispase solution, which allows for easy mechanical separation of the mucosa from the underlying bones and cartilages. The sample is subsequently incubated with 26 U/ml papain solution for 40 min at 37°C with agitation, which delivers fine dissociation of tight and adherens junctions between epithelial cells. This method grants access to a significant number of viable cells in a single-cell suspension for FACS sorting, RNA-seq, and functional assays.

Materials and Reagents

  1. 20, 200, and 1,000 μl Pipette tips (no specific brand)

  2. Thermo ScientificTM screw cap microtubes (2.0 ml) (Fisher Scientific, catalog number: 21-403-202)

  3. 3 ml syringes (BD Biosciences, catalog number: 309657)

  4. 18 G 1.5 in needles (BD Biosciences, catalog number: 305196)

  5. 21 G 1.5 in needles (BD Biosciences, catalog number: 305167)

  6. 50 ml conical tubes (Crystalgen, catalog number: 23-2263)

  7. 12 × 75 mm (5 ml) round bottom polystyrene tubes (Corning, catalog number: 352052)

  8. Petri dish (Falcon, catalog number: 351029)

  9. Aluminum foil

  10. Dispase powder (Gibco, catalog number: 17105041)

  11. ChATBAC-eGFP mice (B6.Cg-Tg (RP23-268L19-EGFP) 2Mik/J) (The Jackson Laboratory, catalog number: 7902)

  12. 200 Proof Ethanol (Koptec, catalog number: V1001)

  13. DNase I (Sigma, catalog number: 10104159001)

  14. Dulbecco’s Phosphate-Buffered Saline (PBS) (Boston BioProducts, catalog number: BSS-220DM-C)

  15. DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) (ThermoFisher Scientific, catalog number: 11320033)

  16. Heat-inactivated fetal bovine serum (FBS) (ThermoFisher Scientific, catalog number: 10082139)

  17. Tyrode’s Solution (with bicarbonate, HEPES, and 0.25% BSA, without calcium) (Boston BioProducts, catalog number: PY-912)

  18. Papain from papaya latex (Sigma, catalog number: P3125)

  19. L-cysteine (Sigma, catalog number: C7352)

  20. Tyrode’s Solution (with HEPES and calcium) (Boston BioProducts, catalog number: BSS-355)

  21. Leupeptin trifluoroacetate salt (leupeptin) (Sigma, catalog number: L2023)

  22. HBSS, 1× without calcium, magnesium, and phenol red (Hank’s Balanced Salt Solution) (Corning, catalog number: 21-022-CV)

  23. EDTA (0.5 M), pH 8.0. RNase-free (Thermo Fisher, catalog number: AM9260G)

  24. TruStain FcXTM (anti-mouse CD16/32) antibody (Biolegend, catalog number: 101320)

  25. Pacific Blue anti-mouse CD45 monoclonal antibody (Biolegend, catalog number: 103126)

  26. Allophycocyanin (APC) anti-mouse CD326 (EpCAM) monoclonal antibody (Biolegend, catalog number: 118214)

  27. Propidium iodide (PI) (Sigma, catalog number: P4170)

  28. 70% EtOH (500 ml) (see Recipes)

  29. Dispase solution (see Recipes)

  30. DMEM-based stopping solution (see Recipes)

  31. Tyrode buffer-based digesting solution with papain (see Recipes)

  32. Tyrode buffer-based stopping solution (see Recipes)

  33. FACS buffer (washing buffer for flow cytometry) (see Recipes)

Equipment

  1. Dissection scissors (straight) (Fisher Scientific, catalog number: 08-951-5)

  2. Forceps (straight, serrated) (Fisher Scientific, catalog number: 13-812-36)

  3. Pipettes (P10, P200, P1000) (no specific brand)

  4. FisherbrandTM IsotempTM General Purpose Deluxe Water Bath (Fisher Scientific, catalog number: FSGPD05)

  5. FisherbrandTM Multiplatform Shaker (Fisher Scientific, catalog number: 88-861-021)

  6. Dissecting microscope (Leica, catalog number: M165FC)

  7. Scalpel (No.10) (ThermoFisher Scientific, catalog number: 3120032)

  8. Fisher Vortex Genie 2 (Fisher Scientific, catalog number: 12-812)

  9. 100 μM FisherbrandTM Sterile Cell Strainers (Fisher Scientific, catalog number: 22-363-549)

  10. Sorvall Legend X1R Centrifuge (ThermoFisher Scientific, catalog number: 75004261)

  11. BD LSRFortessaTM Flow Cytometer (BD Biosciences)

Software

  1. FlowJo v.8 (FlowJo, LLC, https://www.flowjo.com)

  2. Prism 7 (GraphPad Software, https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. 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.


  2. 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 FcXTM (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, CD45low/neg and EpCAMhigh cells constituted 14-18% of all live cells. Brush cells were defined as EpCAMhigh and GFP+ cells, and comprised 14-24% of the EpCAMhigh population 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 SSClow and SSChigh populations based on their size, and represent microvillous cells and solitary chemosensory cells, respectively. SSClow comprised the majority of GFP+ cells (94%), while SSChigh cells 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. CD45low/neg were evaluated on their expression of EpCAM. Brush cells were defined as EpCAMhigh GFP+ cells and were further specified on forward scatter and side scatter characteristics. SSChigh population represents solitary chemosensory cells, and SSClow 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 CD45low/neg cells were evaluated on their expression of EpCAM. C. Number of live cells recovered from nasal mucosa and frequency of EpCAMhigh epithelial cells as percent of live cells. D. Frequency of nasal brush cells presented as percent of live cells, percent of EpCAMhigh population and number of nasal brush cells per mouse nose. E. Frequency of SSChigh (solitary chemosensory cells) and SSClow (microvillous cells) populations as percent of all EpCAMhigh 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

  1. 70% EtOH (500 ml)

    350 ml 200 proof pure ethanol

    150 ml distilled H2O

    Mix well

    This solution is flammable.

  2. 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.

  3. 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

  4. 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

  5. Tyrode buffer-based stopping solution, prepare fresh prior to isolation

    Tyrode’s Solution (with HEPES and calcium)

    2 μl/ml leupeptin (5 mg/ml)

  6. 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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简介

[摘要]孤立的化学感应上皮细胞散布在大多数黏膜表面。它们被称为肠粘膜中的簇状细胞、气管中的刷状细胞以及鼻粘膜中的孤立化学感应细胞和微绒毛细胞。它们是上皮中 IL-25 的主要来源,也参与乙酰胆碱的产生。我们最近证明了鼻孤化学感应(刷)细胞可以响应于刺激钙离子载体产生半胱氨酰白三烯的鲁棒级别,过敏原,和危险相关分子,例如ATP和UTP,并 这种机制取决于嘌呤能受体 P2Y2 的刷状细胞表达。该协议描述了一种有效的小鼠鼻刷细胞分离方法。该方法基于物理分离鼻腔粘膜层并用分散酶预孵育,然后用木瓜蛋白酶溶液消化。通过这种方式获得的单细胞悬浮液含有刷细胞的荧光激活细胞分选(FACS),RNA测序高产率,和离体测定法。

图文摘要:

用于刷细胞分离的鼻消化工作流程。



[背景]刷状细胞是化学感应上皮细胞,以其苦味受体的表达而闻名(Finger等人,2003 年;Krasteva 和 Kummer,2012 年)。刷细胞也可以被称为绒头细胞和在气道(Krasteva和库默尔,2012),胃肠道已被发现(豪伊特等人。,2016) ,和尿上皮(Deckmann等人,2014)。在形态学上,刷状细胞的特征是顶端微绒毛在细胞表面形成簇状突起,延伸至粘膜腔(Rhodin and Dalhamn, 1956 ; Reid et al. , 2005 )。刷状细胞的保护功能与其产生乙酰胆碱密切相关,导致肽能感觉神经纤维的激活(Krasteva等,2011)。我们之前曾报道过刷状细胞来源的 IL-25 在引发肺 2 型炎症和上皮细胞重塑中的作用(Bankova等,2018)。我们最近报道了鼻刷细胞通过嘌呤能受体 P2Y2 响应空气变应原和 ATP 产生的促炎脂质介质称为半胱氨酰白三烯(Ualiyeva等人,2020 年),表明这些细胞在指导免疫反应方面具有强大的功能。气道。
在鼻子中,粘膜衬里由嗅觉和呼吸上皮组成(Adams,1972) 。在小鼠中,嗅觉上皮占整个鼻粘膜的 50%,覆盖在中上鼻甲和鼻中隔的尾部/后部区域,而呼吸上皮覆盖了约 45% 的鼻腔。前部(Gross等人,1982 年;Chamanza 和 Wright,2015 年)。鼻子呼吸道粘膜中的孤立化学感应细胞对刺激物和细菌有反应,并被确立为小鼠(Finger等人,2003 年;Sbarbati 和 Osculati,2003 年)和人类(Lee等人, 2014 年;科汉斯基等人,2018 年)。微绒毛细胞是在嗅觉感觉神经元层上方的顶端嗅觉上皮中发现的相关细胞,并支持支持细胞(Hansen and Finger,2008)。两个微绒毛细胞和孤化学感觉细胞表达聊天,胆碱乙酰转移酶,一个Ë nzyme必需的神经递质乙酰胆碱的合成(Krasteva等人,2011),TRPM5 ,在化学感受细胞中的瞬时受体电位基因普遍存在(佩雷斯等人, 2002;Kaske等人,2007 年;Zhang等人,2007 年;Ogura等人,2011 年;Genovese 和 Tizzano,2018 年),并依赖于Pou2f3 ,一种分化鼻、气管中所有化学感应细胞所需的转录因子,和肠(Ohmoto等人,2013;山口等人,201 4;毛奇。等人,2016 )。
我们最近的研究使用荧光-活化细胞分选(FACS)基于所述荧光报告表达聊天(聊天-EGFP)和大小和粒度特性[前向散射(FSC)和侧向散射(SSC)]使我们能够区分olfactory-来自富含呼吸的孤立化学感应细胞的富集微绒毛细胞。我们通过大量 RNA 测序 (RNA-seq) 证实,这两个群体都属于更大的化学感应簇/刷细胞家族,并且主要以味觉受体的不同表达为特征。该协议描述了我们分离混合鼻化学感应刷细胞群的方法,提供了约 20,000/小鼠的高产量,可以通过 FACS 和 RNA-seq 进一步表征鼻腔中这些细胞类型之间的异同腔。
几个小组报告了从呼吸道鼻黏膜和微绒毛细胞从嗅觉黏膜成功分离孤立的化学感应细胞。Gulbransen ë吨人 (2008)使用补充有 20 U/ml 木瓜蛋白酶的基于 Tyrode 的溶液从 TRPM5-GFP 荧光报告小鼠的呼吸道粘膜中分离出孤立的化学感应细胞。数百个活细胞用于钙通量研究以响应苦味激动剂。林等人。( 2008 )使用含 10 - 30 U/ml 木瓜蛋白酶的林格溶液从前呼吸道上皮细胞中分离出孤立的化学感应细胞,以测量响应气味刺激物的单个细胞中细胞内钙水平的变化。小仓等。(2011)从 ChAT-eGFP 和TRPM5-GFP 小鼠的主要嗅觉上皮细胞中分离出微绒毛细胞,在室温下用 4 U/ml 木瓜蛋白酶处理 3 - 5 分钟,并显示出来自新鲜分离细胞的钙通量增加,以响应 ATP、土壤细菌裂解液,和苯甲地那。虽然没有提供关于使用所述协议回收的孤立化学感应或微绒毛细胞的总产量的信息,但每个使用 <100 个细胞进行钙通量的实时成像。我们测试了相当浓度的木瓜蛋白酶 (26 U/ml) 以从气管中分离刷细胞,发现气管与高剂量分散酶 (16 U/ml) 的预孵育导致分离的刷细胞增加了20 倍与单独用木瓜蛋白酶消化相比的产量(Ualiyeva等,2019)。
在这里,我们描述了一个分步协议,用于从 ChAT-eGFP 荧光转基因小鼠的小鼠鼻子中分离刷细胞,并由Chat指示增强的绿色荧光蛋白表达。胆碱乙酰转移酶在鼻化学感应刷细胞的两个亚群、气管、肠和尿道刷/簇细胞中高度表达,并且与其他上皮细胞相比,在这些化学感应上皮细胞中特别富集。这允许通过流式细胞仪识别荧光胆碱能刷细胞。单细胞溶液制备过程是基于用分散酶溶液对鼻子进行初始孵育,这样可以轻松地将粘膜与下面的骨骼和软骨机械分离。随后将样品与 26 U/ml 木瓜蛋白酶溶液在 37°C 下搅拌孵育40 分钟,从而使上皮细胞之间的紧密和粘附连接精细分离。此方法允许访问的活细胞的数目显著在单细胞悬浮液用于FACS分选,RNA-SEQ ,和功能测定法。

关键字:鼻刷细胞, 胆碱能上皮细胞, 微绒毛细胞, 孤立的化学感应细胞, 苦味感知细胞, 苦味受体, Tuft细胞

 
材料和试剂
 
20、200和 1,000 μl移液器吸头(无特定品牌)
Thermo Scientific TM螺帽微管(2.0 ml)(Fisher Scientific,目录号:21-403-202)
3 ml注射器(BD Biosciences,目录号:309657)
18 G 1.5 针(BD Biosciences,目录号:305196)
21 G 1.5 针(BD Biosciences,目录号:305167)
50 ml锥形管(Crystalgen,目录号:23-2263)
12 × 75 mm(5 ml)圆底聚苯乙烯管(Corning,目录号:352052)
培养皿(Falcon,目录号:351029)
铝箔
分散粉(Gibco,目录号:17105041)
ChAT的BAC -eGFP小鼠(B6.Cg-TG(RP23-268L19-EGFP)2Mik / J)(杰克逊实验室,目录号:7902)
200 Proof Ethanol(Koptec,目录号:V1001)
DNase I(Sigma,目录号:10104159001)
Dulbecco 磷酸盐缓冲盐水(PBS)(Boston BioProducts,目录号:BSS-220DM-C)
DMEM/F-12(Dulbecco's Modified Eagle 培养基/营养混合物 F-12)(ThermoFisher Scientific,目录号:11320033)
热灭活胎牛血清(FBS)(ThermoFisher Scientific,目录号:10082139)
Tyrode 溶液(含碳酸氢盐、HEPES和0.25% BSA,不含钙)(Boston BioProducts,目录号:PY-912)
来自木瓜乳胶的木瓜蛋白酶(Sigma,目录号:P3125)
L-半胱氨酸(Sigma,目录号:C7352)
Tyrode 溶液(含 HEPES和钙)(Boston BioProducts,目录号:BSS-355)
亮肽素三氟乙酸盐(亮肽素)(Sigma,目录号:L2023)
HBSS,1 ×不含钙、镁和酚红(Hank's Balanced Salt Solution)(Corning,目录号:21-022-CV)
EDTA (0.5 M),pH 8.0。无RNase(Thermo Fisher,目录号:AM9260G)
TruStain FCX TM (抗小鼠CD16 / 32)一个ntibody(Biolegend公司,目录号:101320)
Pacific Blue抗小鼠CD45单克隆抗体(Biolegend,目录号:103126)
别藻蓝蛋白(APC)抗小鼠CD326(EpCAM)单克隆抗体(Biolegend,目录号:118214)
碘化丙啶(PI)(Sigma,目录号:P4170)
70% 乙醇(500 毫升)(见食谱)
分散溶液(见配方)
基于 DMEM 的停止解决方案(参见食谱)
含有木瓜蛋白酶的 Tyrode 缓冲液消化溶液(参见食谱)
基于 Tyrode 缓冲液的停止溶液(见配方)
FACS 缓冲液(流式细胞术洗涤缓冲液)(见配方)
 
设备
 
解剖剪刀(直)(Fisher Scientific,目录号:08-951-5)
镊子(直,锯齿)(Fisher Scientific,目录号:13-812-36)
移液器(P10、P200、P1000)(无特定品牌)
Fisherbrand TM Isotemp TM通用豪华水浴锅(Fisher Scientific,目录号:FSGPD05)
Fisherbrand TM Multiplatform Shaker(Fisher Scientific,目录号:88-861-021)
解剖显微镜(Leica,目录号:M165FC)
手术刀(No.10)(ThermoFisher Scientific,目录号:3120032)
Fisher Vortex Genie 2(Fisher Scientific,目录号:12-812)
100 μM Fisherbrand TM无菌细胞过滤器(Fisher Scientific,目录号:22-363-549)
Sorvall Legend X1R Centrifuge(ThermoFisher Scientific,目录号:75004261 )
BD LSRFortessa TM流式细胞仪(BD Biosciences )
 
软件
 
FlowJo v.8 (FlowJo, LLC, https://www.www.flowjo.com )
Prism 7(GraphPad 软件,https ://www.graphpad.com/scientific-software/prism/ )
 
程序
 
小鼠口鼻部的分离
使用的小鼠:ChAT(BAC)-eGFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J),3-6 月龄男女。所有动物护理和程序均经布莱根妇女医院动物护理和使用委员会 (IACUC) 批准。
安乐死老鼠。用 70% EtOH 将皮毛喷在腹部(见配方 1)。用直剪刀做一个中线腹部切口, 打开腹腔, 通过腹主动脉穿刺给小鼠放血。允许鼠标流血 1-2 分钟,以便血液从头部流出,避免/最大限度地减少进一步裂解红细胞的必要性。
通过切断下颌骨并将头部与身体的其余部分分开来斩首鼠标。
使用直剪刀,切断鼻尖和前门牙。去除头部的皮肤并从周围的肌肉和脑组织中释放头骨。
切断颧弓 (图 1A )。取出顶骨。使纵向切口沿着额骨,从顶部(暴露鼻腔图小号1A-1C )。
 
 
图 1. 用分散酶溶液进行第一次消化的鼻部准备的代表性图像。一个。小鼠头部,从周围组织和大脑中释放出来。双侧颧弓需要去除(黑线)。红色虚线代表沿额骨进一步切开的纵向线。乙。沿额骨纵向切口。Ç 。额骨和鼻骨抬起,鼻腔顶部暴露。d 。将鼻子放入装有预热分散酶溶液的 2 毫升管中。
 
鼻上皮细胞分离
预暖750微升的分散酶溶液(见配方2)在37℃水浴中在2ml管5-10分钟。将隔离的鼻子放入溶液中(图 1D ),并在室温下以 100 rpm 的速度在振荡器上孵育 30 分钟。用铝箔盖使管子避光。
加入 750 µl 的基于 DMEM 的冷终止液减少消化(参见配方 3)。将管子放在冰上 2-5 分钟。
从管中取出消化的鼻子,放在解剖显微镜下的培养皿上,上颚朝下。
去除构成鼻腔上壁的鼻骨。现在,您可以看到覆盖有粘膜的鼻腔(图 2A )。通过沿上颚的纵向切口将鼻腔分成左右两半(图 2B )。
 
 
图 2. 从孤立的鼻子鼻黏膜制备的单细胞制剂的代表性图像。一个。鼻腔侧壁示意图,红色表示鼻粘膜覆盖。乙。鼻腔在培养皿上分成左右两半。在解剖显微镜下用手术刀刮掉粘膜。红色轮廓代表要隔离的鼻粘膜区域。Ç 。将刮下的粘膜转移到 2 ml 管中。d 。用 750 µl基于Tyrode 缓冲液的消化液和木瓜蛋白酶冲洗培养皿。乙。停止消化并涡旋样品后,悬浮液用 18 号针头研磨 8-10 次,然后用 21 号针头研磨 15-20 次。˚F 。细胞悬液通过 100 µm 过滤器过滤到 50 ml 锥形管中。
 
用手术刀刮鼻粘膜,直到在解剖显微镜下除骨头外没有组织可见。将分离的粘膜与小的底层骨骼和软骨转移到 2 ml 管中(图 2C )。
用 750 µl含有木瓜蛋白酶的Tyrode 缓冲液消化液冲洗培养皿(参见配方 4),并将冲洗过的液体转移到含有鼻粘膜的管中(图 2D )。
将分离的鼻粘膜在基于 Tyrode 的消化缓冲液中在 37°C 下孵育 40 分钟,以 210 rpm 的速度摇晃。
以 300 rpm 的速度涡旋消化的组织 10 秒。通过添加冷的 Tyrode 缓冲液终止溶液(参见配方 5)并将样品置于冰上来终止消化。
涡流组织在1 ,000rpm下20秒。用连接到 18 号针头的注射器研磨溶液 8-10 次。去除剩余的骨头和软骨,以避免堵塞注射器。将注射器上的针头换成 21 号针头,再研磨 15-20 次(图 2E )。
将悬浮液通过 100 µm 过滤器放入 50 ml 锥形管中(图 2F )。添加 20-25 ml 冷 FACS 缓冲液(参见配方 6)。
在 4°C 下以 350 × g旋转管子15 分钟。检查管中是否有可见颗粒。 在不干扰细胞沉淀的情况下丢弃上清液,将沉淀重悬在 1 ml FACS 缓冲液中,然后转移到 5 ml 聚苯乙烯管中。用 4 ml 的 FACS 缓冲液填充聚苯乙烯管。
在 4°C 下以 350 × g旋转管子10 分钟。弃去上清液,将沉淀重悬在 100 µl FACS 缓冲液中。
要阻断 Fc 受体,请使用 TruStain FcX TM (抗小鼠 CD16/32)抗体预孵育细胞悬液;在 100 µl 体积中,每 10 6 个细胞使用 1.0 µg 抗体,在冰上放置 5-10 分钟。此步骤后无需洗涤。
在冰上用抗体孵育细胞 45 分钟,避光。用于刷细胞鉴定的抗体组包括每100 µl 体积中每 10 6 个细胞0.25 µg 的太平洋蓝抗小鼠 CD45 单克隆抗体和每100 µl 体积中每 10 6 个细胞0.5 µg 的别藻蓝蛋白 (APC) 抗小鼠 EpCAM 单克隆抗体体积。
通过用 1 ml FACS 缓冲液重新悬浮m 来清洗细胞,并添加额外的 3.8-4 ml 冷 FACS 缓冲液。在 4°C 下以 350 × g旋转10 分钟。丢弃上清液,将沉淀重悬在 300 µl 冷 FACS 缓冲液中。
如果不打算在 FACS 机器上运行整个样本,请执行手动细胞计数。如果在机器上收集整个样本,则可以省略计数步骤。
              在 FACS 前加入碘化丙啶 (PI) 5 µg/ml 以维持 2-3 分钟的活力。
 
数据分析
 
在使用此两步协议(见图形摘要)分离鼻粘膜和消化后,用 EpCAM 和 CD45标记细胞悬浮液。细胞是通过FSC-A / SSC-A选通以排除碎片和通过SSC-H / SSC-W和FSC-H / SSC-W来选择单个细胞(图3A )。碘化丙啶(PI)被使用作为一个死细胞排除标记。用这种方法可以获得从 1.2 × 10 6到 3.1 × 10 6 的活细胞(图 3B )。在我们的分析中,CD45低/阴性和 EpCAM高细胞占所有活细胞的 14-18%。刷细胞被定义为 EpCAM high和GFP +细胞,占 EpCAM high群体的14-24%和所有活细胞的 1.3%。这种方法允许每只小鼠分离 16,000-30,000 个 GFP +刷细胞。鼻刷细胞可以根据它们的大小进一步细分为 SSC低和 SSC高群体,分别代表微绒毛细胞和孤立的化学感应细胞。SSC低占大多数 GFP +细胞 (94%),而 SSC高细胞占刷状细胞的 4%。
 
 
图 3. 鼻刷细胞的流式细胞术分析。细胞悬液用针对 EpCAM 和 CD45 的荧光偶联抗体染色,并通过流式细胞术检查。门控策略之前已发布(Ualiyeva等人,2020 年)。A.代表性流式细胞术门控策略。根据前向和侧向散射特征的大小和粒度对细胞进行门控。进一步排除双联体,并根据没有碘化丙啶染色来选择活细胞。CD45低/阴性根据它们的 EpCAM 表达进行评估。刷细胞被定义为 EpCAM高GFP +细胞,并在前向散射和侧向散射特征上进一步指定。SSC高群代表孤立的化学感应细胞,SSC低刷细胞对应于微绒毛细胞。B. C57BL6 小鼠细胞悬液的流式细胞术分析。将样品预选通单活细胞,和CD45低/负对他们的EpCAM表达细胞进行了评价。Ç 。从鼻粘膜中回收的活细胞数量和EpCAM高上皮细胞的频率,以活细胞的百分比表示。D.鼻刷细胞的频率表示为活细胞百分比、EpCAM高种群百分比和每个小鼠鼻子的鼻刷细胞数量。乙。˚F SSC的requency高(孤化学感受细胞)和SSC低(微绒毛细胞)群体,因为所有的EpCAM%的高GFP +细胞计数和。每个点代表一个单独的鼠标。数据来自 3 个独立的实验,每个实验有 2 只小鼠。
 
该协议已成功应用于分离鼻刷细胞以进行 RNA 测序和功能测定(Ualiyeva等,2020)。
 
食谱
 
70% 乙醇(500 毫升)
350 毫升 200 标准纯乙醇
150 毫升蒸馏水 H 2 O
搅拌均匀
该溶液易燃。
分散溶液,在分离前准备新鲜
16 U/ml 分散粉末
Dulbecco 磷酸盐缓冲盐水 (PBS)
蒸馏水 H 2 O
20 微克/毫升 DNase I
准备 1 × PBS
将 16 U/ml 分散酶粉末和 20 μg/ml DNase I 溶解在 1 × PBS 中
将溶液存放在 4℃。在添加鼻子之前在水浴中预热至37°C 。
基于DMEM的终止液(DMEM,5%FBS)(525毫升)中(可被存储为在4长达2个月℃下)
500 ml Dulbecco's Modified Eagle 培养基/营养混合物 F-12 (DMEM)
25 ml 热灭活胎牛血清 (FBS)
在无菌生物安全柜中制备溶液并储存在 4 °C
含有木瓜蛋白酶的 Tyrode 缓冲液消化溶液(分离前准备新鲜)
Tyrode 溶液(含碳酸氢盐、HEPES和0.25% BSA,不含钙)
20 μl/ml 木瓜蛋白酶 (28 U/mg)
10 微升/毫升 L-半胱氨酸 (25 毫克/毫升)
在无菌生物安全柜中制备溶液并储存在 4 °C
基于 Tyrode 缓冲液的终止液,在分离前准备新鲜
Tyrode 溶液(含 HEPES和钙)
2 μl/ml 亮抑酶肽 (5 mg/ml)
FACS 缓冲液(用于流式细胞术的洗涤缓冲液),可在 4°C 下储存长达 1 个月
HBSS,1 ×不含钙、镁和酚红(汉克平衡盐溶液)
2% 热灭活胎牛血清 (FBS)
2 mM 乙二胺四乙酸(EDTA,0.5 M,pH 8.0)
在无菌生物安全柜中制备溶液并储存在 4 °C
 
致谢
 
这个协议是适于与微小修改小号从一个发表先前的研究Ualiyeva等。(2020) 。该方法基于我们实验室使用的先前的气管刷细胞分离方案(Ualiyeva等,2019)。我们感谢布莱根妇女人类免疫学中心 Flow Core 的 Adam Chicoine 在流式细胞仪分选方面提供的帮助。这项工作得到了美国国立卫生研究院 R01 HL120952 (NAB)、R01 AI134989 (NAB)、U19 AI095219 (NAB、LGB)、K08 AI132723 (LGB) 和 T32 AI00730634 (SU) 以及美国过敏学会的支持、哮喘和免疫学 (AAAAI)/美国肺过敏性呼吸系统疾病奖 (NAB),由 AAAAI 基金会教师发展奖 (LGB) 授予,Steven 和 Judy Kaye 青年创新奖 (NAB),由 Joycelyn C. Austen 基金授予女性医师科学家 (LGB) 的职业发展,以及 Vinik 家族 (LGB) 的慷慨捐赠。
 
利益争夺
 
作者声明没有竞争利益。
 
伦理
 
在动物研究中进行的所有程序均由布莱根妇女医院 IACUC 协议 2016N000517 指导。
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ualiyeva, S., Boyd, A. A., Barrett, N. A. and Bankova, L. G. (2021). Isolation of Nasal Brush Cells for Single-cell Preparations. Bio-protocol 11(18): e4163. DOI: 10.21769/BioProtoc.4163.
  2. Ualiyeva, S., Hallen, N., Kanaoka, Y., Ledderose, C., Matsumoto, I., Junger, W. G., Barrett, N. A. and Bankova, L. G. (2020). Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2. Sci Immunol 5(43).
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