Name: isolation and quantitative evaluation of brush cells from mouse tracheas
Uploaded: 2019-06-12T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas at JoVE.com

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>
	<li>Tizzano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+chemosensory+cells+use+bitter+taste+signaling+to+detect+irritants+and+bacterial+signals.">Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (7), 3210-3215 (2010).</li><li>Genovese, F., Tizzano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvillous+cells+in+the+olfactory+epithelium+express+elements+of+the+solitary+chemosensory+cell+transduction+signaling+cascade.">Microvillous cells in the olfactory epithelium express elements of the solitary chemosensory cell transduction signaling cascade.</a> PLoS One. 13 (9), 0202754 (2018).</li><li>Bankova, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cysteinyl+leukotriene+3+receptor+regulates+expansion+of+IL-25-producing+airway+brush+cells+leading+to+type+2+inflammation.">The cysteinyl leukotriene 3 receptor regulates expansion of IL-25-producing airway brush cells leading to type 2 inflammation.</a> Science Immunology. 3 (28), (2018).</li><li>Krasteva, G., Canning, B. J., Papadakis, T., Kummer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+brush+cells+in+the+trachea+mediate+respiratory+responses+to+quorum+sensing+molecules.">Cholinergic brush cells in the trachea mediate respiratory responses to quorum sensing molecules.</a> Life Sciences. 91 (21-22), 992-996 (2012).</li><li>Nadjsombati, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Succinate+by+Intestinal+Tuft+Cells+Triggers+a+Type+2+Innate+Immune+Circuit.">Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.</a> Immunity. 49 (1), 33-41 (2018).</li><li>von Moltke, J., Ji, M., Liang, H. E., Locksley, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuft-cell-derived+IL-25+regulates+an+intestinal+ILC2-epithelial+response+circuit.">Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit.</a> Nature. 529 (7585), 221-225 (2016).</li><li>Deckmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bitter+triggers+acetylcholine+release+from+polymodal+urethral+chemosensory+cells+and+bladder+reflexes.">Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (22), 8287-8292 (2014).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Members+of+Bitter+Taste+Receptor+Cluster+Tas2r143/Tas2r135/Tas2r126+Are+Expressed+in+the+Epithelium+of+Murine+Airways+and+Other+Non-gustatory+Tissues.">Members of Bitter Taste Receptor Cluster Tas2r143/Tas2r135/Tas2r126 Are Expressed in the Epithelium of Murine Airways and Other Non-gustatory Tissues.</a> Frontiers in Physiology. 8, 849 (2017).</li><li>Krasteva, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+chemosensory+cells+in+the+auditory+tube.">Cholinergic chemosensory cells in the auditory tube.</a> Histochemistry and Cell Biology. 137 (4), 483-497 (2012).</li><li>Krasteva, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+chemosensory+cells+in+the+trachea+regulate+breathing.">Cholinergic chemosensory cells in the trachea regulate breathing.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (23), 9478-9483 (2011).</li><li>Montoro, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+airway+epithelial+hierarchy+includes+CFTR-expressing+ionocytes.">A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.</a> Nature. 560 (7718), 319-324 (2018).</li><li>Plasschaert, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+atlas+of+the+airway+epithelium+reveals+the+CFTR-rich+pulmonary+ionocyte.">A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.</a> Nature. 560 (7718), 377-381 (2018).</li><li>Dwyer, D. F., Barrett, N. A., Austen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Genome+Project,+C.+Expression+profiling+of+constitutive+mast+cells+reveals+a+unique+identity+within+the+immune+system.">Immunological Genome Project, C. Expression profiling of constitutive mast cells reveals a unique identity within the immune system.</a> Nature Immunology. 17 (7), 878-887 (2016).</li><li>Howitt, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuft+cells,+taste-chemosensory+cells,+orchestrate+parasite+type+2+immunity+in+the+gut.">Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.</a> Science. 351 (6279), 1329-1333 (2016).</li><li>Bankova, L. G., Dwyer, D. F., Liu, A. Y., Austen, K. F., Gurish, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+mast+cell+progenitors+to+mucosal+mast+cells+during+allergic+pulmonary+inflammation+in+mice.">Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice.</a> Mucosal Immunology. 8 (3), 596-606 (2015).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (31), 12771-12775 (2009).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+stromal+populations+contribute+to+pulmonary+fibrosis+without+evidence+for+epithelial+to+mesenchymal+transition.">Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (52), 1475-1483 (2011).</li><li>Gerbe, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+epithelial+tuft+cells+initiate+type+2+mucosal+immunity+to+helminth+parasites.">Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.</a> Nature. 529 (7585), 226-230 (2016).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+protein+16A+(TMEM16A)+is+a+Ca2+-regulated+Cl-+secretory+channel+in+mouse+airways.">Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl- secretory channel in mouse airways.</a> Journal of Biological Chemistry. 284 (22), 14875-14880 (2009).</li><li>Olsen, J. V., Ong, S. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+cleaves+exclusively+C-terminal+to+arginine+and+lysine+residues.">Trypsin cleaves exclusively C-terminal to arginine and lysine residues.</a> Molecular and Cellular Proteomics. 3 (6), 608-614 (2004).</li><li>Verma, S., Dixit, R., Pandey, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+Proteases:+Modes+of+Activation+and+Future+Prospects+as+Pharmacological+Targets.">Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.</a> Frontiers in Pharmacology. 7, 107 (2016).</li><li>Huettner, J. E., Baughman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+identified+neurons+from+the+visual+cortex+of+postnatal+rats.">Primary culture of identified neurons from the visual cortex of postnatal rats.</a> Journal of Neuroscience. 6 (10), 3044-3060 (1986).</li><li>Kaiser, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociated+neurons+and+glial+cells+derived+from+rat+inferior+colliculi+after+digestion+with+papain.">Dissociated neurons and glial cells derived from rat inferior colliculi after digestion with papain.</a> PLoS One. 8 (12), 80490 (2013).</li><li>Aoyagi, T., Takeuchi, T., Matsuzaki, A., Kawamura, K., Kondo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leupeptins,+new+protease+inhibitors+from+Actinomycetes.">Leupeptins, new protease inhibitors from Actinomycetes.</a> Journal of Antibiotics (Tokyo). 22 (6), 283-286 (1969).</li><li>Kohanski, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solitary+chemosensory+cells+are+a+primary+epithelial+source+of+IL-25+in+patients+with+chronic+rhinosinusitis+with+nasal+polyps.">Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps.</a> The Journal of Allergy and Clinical Immunology. 142 (2), 460-469 (2018).</li><li>Patel, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solitary+chemosensory+cells+producing+interleukin-25+and+group-2+innate+lymphoid+cells+are+enriched+in+chronic+rhinosinusitis+with+nasal+polyps.">Solitary chemosensory cells producing interleukin-25 and group-2 innate lymphoid cells are enriched in chronic rhinosinusitis with nasal polyps.</a> International Forum of Allergy &amp; Rhinology. , (2018).</li><li>Kouzaki, H., O'Grady, S. M., Lawrence, C. B., Kita, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteases+induce+production+of+thymic+stromal+lymphopoietin+by+airway+epithelial+cells+through+protease-activated+receptor-2.">Proteases induce production of thymic stromal lymphopoietin by airway epithelial cells through protease-activated receptor-2.</a> The Journal of Immunology. 183 (2), 1427-1434 (2009).</li><li>Cambier, J. C., Vitetta, E. S., Kettman, J. R., Wetzel, G. M., Uhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+tolerance.+III.+Effect+of+papain-mediated+cleavage+of+cell+surface+IgD+on+tolerance+susceptibility+of+murine+B+cells.">B-cell tolerance. III. Effect of papain-mediated cleavage of cell surface IgD on tolerance susceptibility of murine B cells.</a> The Journal of Experimental Medicine. 146 (1), 107-117 (1977).</li><li>Nishikado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+protease+antigens+cleave+CD123,+the+alpha+subunit+of+murine+IL-3+receptor,+on+basophils+and+suppress+IL-3-mediated+basophil+expansion.">Cysteine protease antigens cleave CD123, the alpha subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.</a> Biochemical and Biophysical Research Communications. 460 (2), 261-266 (2015).</li></ol>

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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	<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>
		</tr>
		<tr height="40">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
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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 ...

Watch this Scientific Journal Video about Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas at JoVE.com

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.

isolation-quantitative-evaluation-brush-cells-from-mouse

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...