We thank Adam Chicoine at the Brigham and Women&#8217;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), and K08 AI132723 (L.G.B), 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.).

Before conducting the following experiments, ensure that all animal care use and protocols are approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accord with the National Research Council&#39;s &#34;Guide for the Care and Use of Laboratory Animals&#34;&#160;(8th&#160;Edition, 2011) and the ARRIVE guidelines.&#160;All procedures described below have been reviewed and approved by the Institutional Animal Care and Use Committee at the Brigham and Women&#39;s Hospi...

<ol>
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The authors have nothing to disclose.

We found that a combination of high-dose dispase treatment for 40 min followed by a short papain treatment (30 min) provides an optimal protocol for tracheal digestion and brush cell isolation. This combination both avoids extensive digestion and produces the highest yield of brush cells, compared to alternate protocols.
While lung digestion to extract hematopoietic cells has classically relied on mild digestive enzymes like collagenase IV15, isolation of epithelial cel...

Brush cells belong to a class of chemosensory epithelial cells characterized by the expression of bitter taste receptors and the taste receptor transduction machinery found in taste bud cells. Unlike taste bud cells, chemosensory epithelial cells are scattered in epithelial surfaces and are referred to as solitary chemosensory cells (SCCs) and microvillous cells in the nasal epithelium1,2, brush cells in the trachea3,4, and tuft cells in the intestine5,6. Epithelial cells expressing bitter taste receptors and the bitter taste transduction machinery are also found in the urethra7,8 and the auditory tube9. Airway brush cells have unique functions in neurogenic and immune airway responses. They are acetylcholine-producing chemosensory cells that evoke protective respiratory reflexes upon activation with bitter compounds and bacterial metabolites like quorum-sensing substances10. Airway brush cells are also the dominant airway epithelial source of IL-25, which regulates aeroallergen-elicited type 2 inflammation in the airways3.
Characterization of the full transcriptome of lower airway brush cells and their response to environmental stimuli has been limited by their low numbers in the tracheal epithelium and very limited numbers beyond the large bronchi10. Techniques used for the isolation of chemosensory cells from the intestinal epithelium have not yielded proportionally high numbers from the trachea, possibly because of the intimate contacts of tracheal brush cells with nerve endings10 or other tissue-specific factors in the respiratory mucosa such as the composition of adherens and tight junction proteins. Recent reports of successful isolation of tracheal brush cells in higher numbers for single cell RNA sequencing analysis employed either a 2 h incubation with papain or an 18 h incubation with pronase11,12. Since longer incubations with digestive enzymes can decrease cell viability and alter the transcriptional profile of cells from digested tissues13, this could bias comparative analysis with other chemosensory epithelial populations.
Here, we report a method for the isolation of tracheal brush cells for RNA sequencing3. Treatment of the trachea with high-dose dispase separates the epithelium from the submucosa. Subsequent digestion of the epithelial sheet with papain allows for excellent recovery of this structural cell.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Anti-GFP (Polyclonal goat Ig)</td>
			<td style="width:101px;">Abcam</td>
			<td>cat# ab5450</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">APC anti-mouse CD326 (EpCAM)&#160; (G8.8)</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#118214</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">APC Rat IgG2a, k isotype control</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#400511</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">DAPI</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#422801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:345px;">Donkey anti-goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:101px;">Life Technologies/Molecular Probes</td>
			<td>cat#A-11055</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Normal Goat IgG</td>
			<td style="width:101px;">R&#38;D Systems</td>
			<td style="width:136px;">cat#AB-108-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Pacific Blue anti-mouse CD45 (30F-11)</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#103126</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Pacific Blue Rat IgG2b, k isotype control</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#400627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">TruStain FcX (anti-mouse CD16/32) Antibody</td>
			<td style="width:101px;">Biolegend</td>
			<td style="width:136px;">cat#101320</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td>Chemicals, Peptides, and Recombinant Proteins</td>
			<td style="width:167px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:345px;">Dispase</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:136px;">cat# 17105041&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">DNase I</td>
			<td style="width:101px;">Sigma&#160;</td>
			<td>cat# 10104159001</td>
			<td></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:345px;">HEPES-Tyrode&#8217;s Buffer Without Calcium (10 mM HEPES, 135 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM NaH2PO4, 0.25% BSA, 5.5 mM Glucose. Prepared in 18.2 megohms water and filtered through 0.22 &#181;m filter</td>
			<td style="width:101px;">Boston BioProducts</td>
			<td style="width:136px;">cat# PY-912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:345px;">Tyrode&#8217;s Solution (HEPES-Buffered) 140 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2 and 10 mM glucose. Prepared in 18.2 megohms water and filtered through 0.22 &#181;m filter. )</td>
			<td style="width:101px;">Boston BioProducts</td>
			<td style="width:136px;">cat# BSS-355</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">L-Cysteine</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:136px;">cat# C7352</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Leupeptin trifluoroacetate salt</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:136px;">cat# L2023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Papain from papaya latex</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:136px;">cat# P3125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Propidium iodide&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:136px;">cat# P4170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Experimental Models: Organisms/Strains</td>
			<td style="width:101px;"></td>
			<td style="width:136px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">ChATBAC-eGFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J)</td>
			<td style="width:101px;">The Jackson Laboratory</td>
			<td style="width:136px;">7902</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Equipment</td>
			<td style="width:101px;"></td>
			<td style="width:136px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">LSM 800 with Airyscan confocal system&#160;on a&#160;Zeiss Axio Observer Z1 Inverted Microscope</td>
			<td style="width:101px;">Zeiss</td>
			<td style="width:136px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRFortessa</td>
			<td>BD</td>
			<td style="width:136px;">647465</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Disposable equipment</td>
			<td style="width:101px;"></td>
			<td style="width:136px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:345px;">1.5 mL sterile tubes</td>
			<td style="width:101px;">Thomas Scientific</td>
			<td style="width:136px;">1157C86</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">5 mL Poysterene Round-bottom Tube, 12 x 75 mm style</td>
			<td style="width:101px;">Falcon</td>
			<td style="width:136px;">14-959-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">50 mL Polypropylene conical tube, 30 x 115 mm style</td>
			<td style="width:101px;">Falcon</td>
			<td style="width:136px;">352098</td>
			<td></td>
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			<td height="40" style="height:40px;width:345px;">Feather Disposable Scalpel no.12</td>
			<td style="width:101px;">Fisher Scientific</td>
			<td style="width:136px;">NC9999403</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Petri dish, 100 x 15 mm Style</td>
			<td style="width:101px;">Falcon</td>
			<td style="width:136px;">351029</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Sterile cell strainer, 100 &#956;m</td>
			<td style="width:101px;">Fisherbrand</td>
			<td style="width:136px;">cat#22363549</td>
			<td></td>
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isolation and quantitative evaluation of brush cells from mouse tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

This procedure has been successfully implemented to isolate tracheal brush cells for RNA sequencing3. After isolation of the trachea and digestion of the tissue with a 2-step protocol (Figure 1), cells were collected and stained with fluorescently-labeled CD45 and EpCAM after exclusion of dead cells with PI. After gating out doublets based on forward and side scatter characteristics, we defined brush cells as low/negative for CD45, positive for EpCAM and positive for ...

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Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

We developed a procedure for isolation of tracheal brush cells, rare specialized chemosensory epithelial cells from choline acetyltransferase fluorescent reporter mice. This method is based on an initial separation of tracheal epithelium from the submucosa allowing for a subsequent shorter incubation of the epithelial sheet with papain. This allows for robust recovery of tracheal brush cells and has been successfully used for isolation and transcriptional analysis of brush cells by RNA sequencing.Long incubation with digestive enzymes can decrease cell viability and alter the transcriptional profile of cells isolated from tissues. Here we separate the tracheal epithelium with dispase and subsequently incubate it with papain for a short amount of time producing high yields of viable brush cells. Our method can contribute to studies of upper airway epithelium.The same protocol can be applied to isolating brush cells from other mucosal sites such as nasal tissue. Demonstration of our highly reproducible technique will allow peer scientists to isolate and further investigate tracheal brush cells. To begin this procedure, add dispase and DNase I to PBS at the final concentrations shown here to prepare the dispase digestion solution.Make sure that the dispase powder is fully dissolved before warming up the solution in a water bath at 37 degrees Celsius. Next, add 5%heat-inactivated FBS to DMEM to prepare a stopping solution. To prepare the Tyrode I buffer, add papain and L-cysteine to HEPES Tyrode's buffer without calcium.To prepare the Tyrode II buffer, add leupeptin to HEPES Tyrode's buffer without calcium at a concentration of two microliters per milliliter. To prepare the FACS buffer, use Hank's Balanced Salt Solution without calcium, magnesium and phenol red supplemented with two millimolar EDTA and 2%FBS. After euthanizing the mouse, use 21 gauge needles to fix it on a surgical board in the supine position with extended upper and lower extremities.Spray the fur with 70%ethanol to sanitize the area. Using straight forceps, lift the skin and fur of the abdomen and use dissecting scissors to make an incision in the center. Using the scissors, separate the skin from the subcutaneous tissue from the abdomen to the mandibula.While holding the subcutaneous tissue up with the forceps, make a small incision with the scissors in the center of the abdominal wall. Then open the peritoneum with a V-shaped incision. Using the forceps, gently move the small intestine to the side.Locate the abdominal aorta and vena cava and make an incision with the dissecting scissors to allow for rapid exsanguination. Locate the diaphragm. Using an 18 gauge needle, make an opening in the diaphragm just below the sternum to deflate the lungs.Using sharp pointed straight dissecting scissors, cut along the base of the ribs to carefully separate the diaphragm from the ribcage. Use the forceps to lift the exposed end of the sternum and cut the sternum longitudinally from the base of the ribcage to the neck. Use short straight scissors to make a central cervical incision and separate the two lobes of the submandibular gland.After this, use a pair of fine point high precision forceps to carefully remove the surrounding connective tissue and the thymus overlying the carina. Dissect the trachea free by first separating the proximal end at the level of the epiglottis and then dissecting the distal end at the level of the bifurcation of the trachea. Locate the epiglottis and cut the trachea longitudinally from the epiglottis to the carina.To begin, place the trachea into a 1.5 milliliter tube containing 750 microliters of dispase digestion solution pre-warmed to 37 degrees Celsius. Cover the tube with aluminum foil to reduce the exposure to direct light and incubate on a shaker at 200 RPM and at room temperature for 40 minutes. Then add 750 microliters of DMEM with 5%FBS to stop the reaction and place the tube on ice.Transfer the trachea to a Petri dish. Orient the trachea with the epithelial side facing up. The longitudinally dissected trachea has a semi-cylindrical shape maintained by the cartilaginous rings.The epithelium is on the concave surface. Using straight forceps, tether the epiglottis area of the trachea to the Petri dish and use a size 22 disposal scalpel to scrape the epithelium off the trachea. The epithelial layer will separate as a translucent sheet.Use the scalpel to mince the epithelium and transfer the epithelial layer to a two milliliter tube. Rinse the Petri dish with 750 microliters of Tyrode I buffer and transfer this to the tube containing the epithelial layer. Cover the tube with aluminum foil to reduce the exposure to direct light and incubate at 37 degrees Celsius on a shaker at 200 RPM for 30 minutes.Then add 750 microliters of cold Tyrode II buffer. Vortex the digested tissue vigorously for 20 to 30 seconds. Using a syringe attached to an 18 gauge needle, triturate the homogenate 10 times.After this, switch to a 21 gauge needle and triturate 10 to 20 more times. Filter the cells through a 100 micrometer strainer into a 50 milliliter conical tube. Add cold FACS buffer at a volumetric ratio of 30 to one.Centrifuge at 350 times g and at four degrees Celsius for 10 minutes. Discard the supernatant and resuspend the pellet in cold FACS buffer. Transfer this suspension to a 12 by 75 millimeter polystyrene tube.Centrifuge again at 350 times g and at four degrees Celsius for 10 minutes. Discard the supernatant and resuspend the pellet in 100 microliters of FACS buffer. Next, add one microliter of anti-mouse CD1632 blocking antibody to block non-specific binding and incubate on ice for 15 minutes.Then add antibodies in the respective isotype controls as outlined in the text protocol. Incubate on ice for 45 minutes while protected from direct light. After this, add 4.5 milliliters of cold FACS buffer and mix.Centrifuge at 350 times g and at four degrees Celsius. Discard the supernatant and resuspend the pellet in 300 microliters of cold FACS buffer. Add propidium iodide immediately before flow cytometric sorting.In this study, tracheal brush cells from ChAT eGFP acetylcholine fluorescent reporter mice are successfully isolated for RNA sequencing. Cells are identified from debris by forward and side scatter angle. Doublets are excluded using forward scatter height and width and side scatter height and width.The doublets are the cells that have high width values. Within the single cells, the live cells are identified as the population that is propidium iodide negative. Within the live single cells, the CD45 low to negative cells are identified based on the isotype control.Within the CD45 low negative cells, the EpCAM positive cells that are also eGFP positive in the FITC channel are the population of brush cells. Brush cell numbers are altered by exposure to microbial metabolites and protozoa and therefore reflect the variability of institutional microbiota. Therefore, we would suggest estimating the number of brush cells by fluorescence microscopy to gauge the expected number of isolated brush cells by flow cytometric sorting.Make sure to properly separate the epithelial layer of the trachea as it determines the yields of isolated brush cells. The isolated tracheal brush cells can be used in a broad array of assays such as RNA sequencing, cell culture and functional analysis.

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Research

JoVE Journal

Immunology and Infection

7.1K Görüntüleme.  Harvard Medical School. Biz trakeal fırça hücrelerinin izolasyon u için bir prosedür geliştirdi, kolin asetiltransferaz floresan muhabir farelerden nadir özel kemosensory epitel hücreleri. Bu yöntem, trakeal epitelin submukozadan ilk olarak ayrılmasına dayanır ve epitel tabakasının papain ile daha kısa sürede inkübasasına olanak sağlar. Bu, trakeal fırça hücrelerinin sağlam bir şekilde kurtarılmasını sağlar ve Fırça hücrelerinin RNA dizilimi ile izolasyon ve transkripsiyonel analizi i?...