This work is supported by grants from the Canadian Institutes of Health Research (178918), Fonds de recherche en sant&#233; du Qu&#233;bec &#8211; Vision Health Research Network.

NOTE: Male C57BL/6 mice (5&#8211;7 weeks old) are from Charles River Laboratories International, Inc. (St-Constant, Quebec, Canada.) and manipulated according to protocols approved by the CHU Sainte-Justine Animal Care Committee. Mouse bronchial tissue explants provide a good source of primary bronchial epithelial cells for investigating the proapoptotic effects of LMPs on epithelial cells. This protocol describes the in vitro generation of LMPs, as well as a method for detecting apoptotic epithelial cells on LMPs-treated bronchial tissue explants. This protocol consists of 3 sections.
1. LMPs Production and Characterization
NOTE: To prevent contamination, ensure that all materials used in this experiment are sterile or autoclaved. Perform all steps at RT in a biological safety cabinet under sterile condition, unless otherwise indicated.
1.1) Stimulation and Collection of MPs9
<ol>
	<li>Thaw an aliquot of 10 million CEM T cells in a 37 &#176;C water bath. Dilute in 10 ml pre-warmed serum-free hematopoietic medium such as X-VIVO, in a 15 ml sterile tube and centrifuge at 200 g x 5 min. Aspirate the supernatant and resuspend cells in 5 ml pre-warmed medium.</li>
	<li>Transfer cells into a T75 tissue culture flask (for suspension cells) with 15 ml pre-warmed hematopoietic medium such as X-VIVO and incubate for 4 days in a humidified incubator at 37 &#176;C with 5% CO2 .</li>
	<li>After 4 days, transfer all the culture medium and cells into a T175 tissue culture flask containing 100 ml fresh medium. Continue incubating the cells for about 72 hr under the same conditions until they have grown to a density of 2 million cells/ml.</li>
	<li>Evenly split cells between four T175 flasks each containing 150 ml fresh medium and continue cell culture until cells have grown (approximately 48 hr incubation) to a density of 2 million/ml.</li>
	<li>Collect cells from each flask by centrifugation at 200 x g for 5 min and resuspend 300&#160;x 106&#160;cells into a new T175 flask containing 150 ml fresh medium, to maintain the 2 million/ml cell density .</li>
	<li>Add actinomycin&#160;D (dissolved in DMSO at 2 mg/ml) to the medium at a final concentration of 0.5 &#181;g/ml and incubate for 24 hr.</li>
	<li>Transfer all the culture medium into 50 ml conical tubes and spin down the cells at 750 x g for 5 min. Transfer the supernatant into 50 ml conical tubes and centrifuge at 1,500 x g for 15 min to remove large cell fragments.</li>
	<li>Transfer the supernatant into a 250 ml bottle and ultracentrifuge at 12,000 x g for 50 min. Discard the supernatant and collect pellets.</li>
	<li>Wash LMPs-enriched pellets with 40 ml sterile PBS in a 50 ml tube by centrifugation at 12,000 x g for 50 min. Repeat this step twice.</li>
	<li>Collect the last wash supernatant; it will be used as vehicle control. Suspend the LMPs pellets in 1 ml of PBS and transfer into a 1.5 ml sterile microtube. Aliquot and store isolated LMPs at -80 &#176;C (to avoid multiple free-thaw cycles).</li>
</ol>
1.2) Characterization of MPs via FACS Analysis 4
<ol>
	<li>Prepare 2 samples of annexin buffer, 1 with and another without CaCl2: Hepes 10 mM, NaCl 140 mM, plus or minus 5 mM CaCl2.</li>
	<li>Filter annexin buffer and FACS flow sheath fluid using a 0.22 &#181;m filter to remove particles.</li>
	<li>Dilute 1 &#181;l of LMPs in 44 &#181;l of annexin buffer with 5 mM CaCl2 into a FACS tube. Prepare another tube with 1 &#181;l of LMPs in 44 &#181;l of annexin buffer without CaCl2 (negative control).</li>
	<li>Add 5 &#181;l of annexinV-Cy5 in each tube and mix well. Incubate for 15 min at RT in the dark. Stop the reaction by diluting the mix with 400 &#181;l of FACS flow sheath fluid in each tube.</li>
	<li>Add 10 &#181;l (200,000 beads) of 7 &#181;m counting beads suspension as an internal standard in each tube to obtain an absolute count.</li>
	<li>Establish gates of relative size (FSC-H, PMT E00, log scale) and relative granularity (SSC-H, PMT 325, log scale) dot plot on the flow cytometer using size-calibrated fluorescent beads of 1 &#181;m (gate 1) and counting beads gate at 7 &#181;m (gate 2).</li>
	<li>Analyze the LMPs sample on FSC-H /SSC-H plot using the established gates and FL-4 channel for annexin (PMT 765, log scale) dot plot, by acquiring a signal until 20,000 counting beads are reached in gate 2.</li>
	<li>Determine the positive annexinV events of LMPs in annexin buffer containing CaCl2, and then subtract the events of LMPs in annexin buffer without CaCl2 (negative control).</li>
	<li>Calculate the absolute number of MPs based on the following equation: 
	<img alt="Equation 1" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li>
</ol>
1.3) Determination of MP Protein Concentration (Bradford Assay)
<ol>
	<li>Prepare 5 serial dilutions of a protein standard from 1.25 to 20 &#181;g/ml. Pipette 800 &#181;l of each standard and sample solution into a clean test tube in duplicate. Add 200 &#181;l of Bradford dye reagent to each tube. Mix well, then incubate at RT for 5 min.</li>
	<li>Measure absorbance at 595 nm. Determine the protein concentration of LMPs using the linear regression of standard curve.</li>
</ol>
2. Bronchial Tissue Explants and LMPs Treatment
NOTE: Pay special attention to the sterile working environment, and aseptically prepare the solutions and medium used in following experiments. To prepare the Complete Healing Medium, add 1 ml of Tissue Healing Medium Supplements with Serum (thawed on ice) to 100 ml Tissue Healing Medium and mix well.
2.1) Preparation of Bronchial Tissue Explants
<ol>
	<li>Before culturing, scratch 6 areas of 1 cm2 each at the edge of the surface of each 100 mm tissue culture dish with a scalpel blade. Coat each scratched 100 mm tissue culture dish with 2 ml of the culture dish coating solution, and incubate the dish in a humidified CO2 incubator O/N at 37 &#176;C. Vacuum aspirate the surplus solution and fill the dish with 15 ml of Tissue Washing Medium.</li>
	<li>Euthanize C57BL/6 mice (5 to 7 weeks old) by CO2 inhalation according to protocols approved by the animal care ethics committee.</li>
	<li>Aseptically dissect lung tissue with scalpel, Dumont super fine tweezer, and surgical scissors. Carefully remove parenchyma and blood vessels. Place lung tissue into ice-cold Tissue Washing Medium for transport to the laboratory, if applicable.</li>
	<li>Further dissect bronchus submerged in the Tissue Washing Medium and separate the bronchus with a diameter of 1 to 2.5 mm from peripheral lung tissues. Slice bronchial tissues into ~5 mm thick bronchial rings with a scalpel.</li>
	<li>Use a scooping motion with the sterile curved microdissecting forceps to pick up the bronchial fragments and place them onto the scratched areas of the dishes.</li>
	<li>Remove the Tissue Washing Medium, and incubate the fragments at RT for ~5 min to allow them to adhere to the dishes.</li>
	<li>Add 10 ml of Complete Healing Medium to each dish and place them in a controlled atmosphere modular incubator chamber. Flush the chamber with high O2 gas mixture (70% O2, 25% N2 and 5% CO2,). Place the chamber in a benchtop orbital incubator and shake it at 37 &#176;C. Shake the chamber for 24 hr at 10 cycles per minute to allow the medium to flow intermittently over the fragments.</li>
	<li>After 24 hr incubation, observe the tissue explants under a phase-contrast inverted light microscope. Select bronchial explants with complete, fine hair movement and lively bronchial epithelium for subsequent LMPs treatment.</li>
</ol>
2.2) LMPs Treatment
<ol>
	<li>Prepare complete growth medium as follows: thaw growth medium supplements with serum and fibroblast inhibitor on ice. Add 1 ml of the growth medium supplements with serum and 200 &#956;l fibroblast inhibitor to 100 ml of growth medium; mix thoroughly. Warm the complete growth medium at 37 &#176;C for 10 min prior to use.</li>
	<li>Dilute isolated LMPs in a new sterile eppendorf tube with PBS to prepare a LMPs stock at a concentration of 800 &#181;g/ml.</li>
	<li>Add 0.5 ml of Complete Growth Medium to each well of a 12-well tissue culture plate.</li>
	<li>Transfer the selected bronchial explants with the curved microdissecting forceps from the previous protocol (section 2.1) to each well of the tissue culture plate.</li>
	<li>Label the culture plate appropriately to identify LMPs treatment wells and control wells. Add 25 &#181;l LMPs stock into each LMPs treatment well (for a final concentration of 40 &#956;g/ml) and 25 &#181;l control vehicle (see LMPs production) to the control wells.</li>
	<li>Continue the incubation in a controlled atmosphere modular incubator chamber at 37 &#176;C with gentle shaking.</li>
	<li>After 24 hr, wash explants 3 times with PBS and proceed to the next step (4% paraformaldehyde [PFA] fixation).</li>
</ol>
3. Histopathological Examination
3.1) Prepare the Following Solutions Before Proceeding to the Next Steps
<ol>
	<li>Prepare 1x PBS buffer by mixing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4.</li>
	<li>To prepare 4% PFA, dissolve 20 g of PFA in 400 ml of water, heated at 60 &#176;C with stirring; add a few drops of 10 M NaOH to clear the solution. Next add 1x PBS buffer and adjust the volume to 500 ml and pH to 7.4. Filter and aliquot; store at -20 &#176;C.</li>
	<li>Prepare the following dehydration or rehydration reagents; 100%, 90%, 70%, 50% ethanol and xylene.</li>
</ol>
3.2) Explant Fixation and Tissue Section Deparaffinization
<ol>
	<li>Place each explant in a labelled microcentrifuge tube with 1.5 ml of 4% PFA and incubate O/N at 4 &#176;C. Rinse the explants twice with 1x PBS.</li>
	<li>Dehydrate explants through an alcohol series (70% ethanol: 3 times 30 min each; 90% ethanol: 2 times 30 min each; 100% ethanol: 3 times 30 min each; then xylene: 3 times 20 min each). Perform all steps at RT in a fume hood.</li>
	<li>Imbed tissue explants in paraffin at 58 &#176;C in an oven. Prepare 5 &#181;m thick tissue sections using a rotary microtome.</li>
	<li>Float the sections in a 56 &#176;C water bath, and then mount the sections onto labeled histological slides. Place the slides in manual staining racks and dry at 65 &#176;C for 1 hr. Allow the slides to cool at RT.</li>
	<li>Dip the racks in 4 consecutive stain dishes containing xylene for 10 min each to remove paraffin. Dip the racks in an ethanol series to remove xylene: 100%, then 95%, then 80%, then 70%, then 50% ethanol (5 min for every step). Rinse the racks with tap water for 5 min to remove ethanol.</li>
</ol>
3.3) Hematoxylin and Eosin (H&#38;E) Staining&#160;
<ol>
	<li>Continue working with the fixed tissue sections; place the rack into a staining dish filled with Mayer&#39;s Hematoxylin for 15 min. Rinse the rack with tap water to remove Hematoxylin for 20 min.</li>
	<li>Place in distilled water for 30 sec.Place in 95% ethanol for 30 sec. Place in Eosin Y solution staining dish for 1 min. Dehydrate through 2 changes of 95% ethanol, 100% ethanol, and xylene for 2 min each.</li>
	<li>Perform a quick check under a microscope to ensure that excess eosin is removed. Place 2 to 3 drops of Mounting Medium (Fisher SP15-100) onto each slide, then cover with a cover glass.</li>
</ol>
3.4) In Situ Cell Death Detection: TUNEL Assay
<ol>
	<li>Before beginning, prepare proteinase K working solution: 20 &#181;g/ml in 10 mM Tris/HCl, pH 7.4.</li>
	<li>Repeat steps 1 to 5 of section 3.2 (Explant fixation and tissue section deparaffinization). Rinse the slides with deionized H2O.</li>
	<li>Immerse the slides with 1x PBS for 10 min. Drain the excess PBS. Incubate tissue sections for 30 min at RT&#160;with proteinase K working solution. Rinse slides twice with 1x PBS.</li>
	<li>Perform the TUNEL assay as described in the Instruction Manual of cell death detection kit. Mount using mounting medium, and coverslip manually with glass coverslips.</li>
	<li>Analyze samples under a light microscope. Use Image Pro 4.5 to analyze the apoptotic cells in brown color.</li>
</ol>

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	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., 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=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, 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=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, 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=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. 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F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+protagonists+of+a+novel+communication+network+for+intercellular+information+exchange.">Microparticles: protagonists of a novel communication network for intercellular information exchange.</a> Circ Res. 107 (9), 1047-1057 (2010).</li><li>Yang, C., 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=Anti-proliferative+and+anti-tumour+effects+of+lymphocyte-derived+microparticles+are+neither+species-+nor+tumour-type+specific.">Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.</a> J Extracell Vesicles. 3, (2014).</li><li>Soleti, 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=Microparticles+harboring+Sonic+Hedgehog+promote+angiogenesis+through+the+upregulation+of+adhesion+proteins+and+proangiogenic+factors.">Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.</a> Carcinogenesis. 30 (4), 580-588 (2009).</li></ol>

The authors have nothing to disclose.

MPs are active mediators of intercellular cross talk and their study is promising in many areas of science.11 This study presented a detailed protocol for in vitro large-scale generation of LMPs derived from an apoptotic T cell line. These MPs express a large repertoire of lymphocyte molecules and are biologically implicated in the regulation of cellular and tissue homeostasis. However, LMPs derived from different sources may be biologically different.4,9,12,13
L...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table><tbody><tr><td>LMPs production and characterization</td><td></td><td></td><td></td></tr><tr><td>CEM T cells&amp;nbsp;</td><td>ATCC&amp;nbsp;</td><td>CCL-119</td><td></td></tr><tr><td>X-VIVO 15 medium&amp;nbsp;</td><td>Cambrex, Walkersville</td><td>04-744Q</td><td></td></tr><tr><td>Flask T75</td><td>Sarstedt</td><td>83.1813.502</td><td></td></tr><tr><td>Flask T175</td><td>Sarstedt</td><td>83.1812.502</td><td></td></tr><tr><td>Actinomycin&amp;nbsp;D&amp;nbsp;</td><td>Sigma Chemical Co.</td><td>A9415-2mg</td><td></td></tr><tr><td>PBS</td><td>Lifetechnologies</td><td>14190-144</td><td></td></tr><tr><td>0.22 &amp;micro;m filter</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Annexin-VCy5</td><td>BD Pharmagen&amp;nbsp;</td><td>559933</td><td></td></tr><tr><td>FACS flow solution</td><td>BD Bio-sciences</td><td>342003</td><td></td></tr><tr><td>Fluorescent microbeads (1 &amp;micro;m)</td><td>Molecular Probes&amp;nbsp;</td><td>T8880</td><td></td></tr><tr><td>Polysterene counting beads (7 &amp;micro;m)</td><td>Bangs laboratories</td><td>PS06N/6994</td><td></td></tr><tr><td>Polypropylene FACS tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>1 ml pipet</td><td>Fisher</td><td>13-678-11B</td><td></td></tr><tr><td>5 ml pipet</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>25 ml pipet</td><td>Ultident</td><td>DL-357551</td><td></td></tr><tr><td>1.5 ml conical polypropylene micro tube</td><td>Sarstedt</td><td>72.690</td><td></td></tr><tr><td>15 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.554.205</td><td></td></tr><tr><td>50 ml conical polypropylene tube</td><td>Sarstedt</td><td>62.547.205</td><td></td></tr><tr><td>50 ml high speed polypropylene copolymer tube</td><td>Nalgene</td><td>3119-0050</td><td></td></tr><tr><td>250 ml high speed polypropylene bottle</td><td>Beckman</td><td>356011</td><td></td></tr><tr><td>Protein assay (Bradford assay)</td><td>Bio-Rad Laboratories</td><td>500-0006</td><td></td></tr><tr><td>Protein assay standard II</td><td>Bio-Rad Laboratories</td><td>500-0007</td><td></td></tr><tr><td>Test tube 16 x 100</td><td>VWR</td><td>47729-576</td><td></td></tr><tr><td>Test tube 12 x 75</td><td>Ultident</td><td>170-14100005B</td><td></td></tr><tr><td>Cell incubator&amp;nbsp;</td><td>Mandel</td><td>Heracell 150</td><td></td></tr><tr><td>Low speed centrifuge</td><td>IEC</td><td>Centra8R</td><td></td></tr><tr><td>High speed centrifuge</td><td>Beckman</td><td>Avanti J8</td><td></td></tr><tr><td>High speed rotor for 250ml bottle</td><td>Beckman</td><td>JLA16.250</td><td></td></tr><tr><td>High speed rotor for 50ml tube</td><td>Beckman</td><td>JA30.50</td><td></td></tr><tr><td>Fow cytometry&amp;nbsp;</td><td>BD Bio-sciences</td><td>FACS Calibur</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman</td><td>Series 600</td><td></td></tr><tr><td>Bronchial tissue explants and sections&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6 mice (5-7 weeks old)&amp;nbsp;&amp;nbsp;</td><td>Charles River Laboratories, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Mouse Airway PrimaCell&amp;trade; System:</td><td>CHI Scientific, Inc.</td><td>2-82001</td><td></td></tr><tr><td>&amp;nbsp;Rib-Back Carbon Steel Scalpel Blades</td><td>Becton Dickinson AcuteCare</td><td>371310</td><td>#10</td></tr><tr><td>Scalpel Handle</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>10003-12</td><td>#7</td></tr><tr><td>phase-contrast inverted microscope</td><td>Olympus Optical CO., LTD.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>CK2</td><td></td></tr><tr><td>high O&lt;sub&gt;2&lt;/sub&gt; gas mixture&amp;nbsp;</td><td>VitalAire Canada Inc.</td><td></td><td></td></tr><tr><td>modular incubator chamber</td><td>Billups-Rothenberg Inc.</td><td>MIC-101</td><td></td></tr><tr><td>MaxQ 4000 incubated orbital shaker</td><td>Barnstead Lab-Line,&amp;nbsp;</td><td>SHKA4000-7</td><td></td></tr><tr><td>12-well tissue culture plate</td><td>Becton Dickinson and Company</td><td>353043</td><td></td></tr><tr><td>Plastic tissue culture dishes (100 mm)</td><td>Sarstedt, Inc.</td><td>83.1802</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>14060-09</td><td>Straight, sharp, 9cm longth</td></tr><tr><td>Half-curved Graefe forceps</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>11052-10</td><td></td></tr><tr><td>humidified CO2 incubator</td><td>Mandel Scientific Company Inc.&amp;nbsp;</td><td>SVH-51023421</td><td></td></tr><tr><td>&amp;nbsp;Histopathological examination&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>formalin formaldehyde</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT5011</td><td></td></tr><tr><td>paraffin</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>T555</td><td></td></tr><tr><td>ethyl alcohol</td><td>Merck KGaA, Darmstadt</td><td>EX0278-1</td><td></td></tr><tr><td>&amp;nbsp;glutaraldehyde&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>G6403</td><td></td></tr><tr><td>Cacodylate</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>31533</td><td></td></tr><tr><td>microscope slides</td><td>VWR Scientific Inc.&amp;nbsp;</td><td>48300-025</td><td>25x75 mm</td></tr><tr><td>Xylene</td><td>Fisher scientific&amp;nbsp; International, Inc.</td><td>X5-4</td><td></td></tr><tr><td>Mayer&#039;s hematoxylin</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>MHS16</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>HCl&amp;nbsp;</td><td>Fisher scientific&amp;nbsp; International, Inc.&amp;nbsp;&amp;nbsp;</td><td>A144s-500</td><td></td></tr><tr><td>eosin&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td>HT110116</td><td>Funnel with filter paper&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Permount&amp;trade; Mounting Medium</td><td>Thermo Fisher Scientific Inc.&amp;nbsp;</td><td>SP15-100</td><td></td></tr><tr><td>glass coverslip</td><td>surgipath medical industries, Inc.</td><td>84503</td><td>24&amp;times;24&amp;nbsp;#1&amp;nbsp;</td></tr><tr><td>TUNEL detection kit</td><td>In Situ Cell Death Detection, POD</td><td>11 684 817 910</td><td></td></tr><tr><td>oven</td><td>Despatch Industries Inc.</td><td>LEB-1-20</td><td></td></tr><tr><td>rotary Microtome</td><td>Leica Microsystems Inc.</td><td>RM2145</td><td></td></tr><tr><td>filter paper</td><td>Whatman International Ltd.</td><td>1003150</td><td>#3</td></tr><tr><td>Microscope</td><td>Nikon Imaging Japan Inc.</td><td>E800</td><td></td></tr><tr><td>staining dish complete</td><td>Wheaton Industries, Inc.</td><td>900200</td><td>including dish, rack, cover</td></tr><tr><td>1.5 ml eppendorf tube</td><td>Sarstedt Inc.&amp;nbsp;</td><td>72.69</td><td>39x10 mm</td></tr><tr><td>Orbital and Reciprocating Water Bath</td><td>ExpotechUSA</td><td>ORS200</td><td></td></tr><tr><td>phosphate buffered saline&amp;nbsp;&amp;nbsp;</td><td>GIBCO</td><td>14190-144</td><td></td></tr><tr><td>fume hood</td><td>Nicram RD Service</td><td>3707E</td><td></td></tr></tbody></table>

generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

LMPs were characterized with annexin V staining10 by fluorescence-activated cell sorting (FACS) analysis and gated using 1 &#181;m beads in which 97% of MPs (&#8804;1 &#181;m) were annexin-V-Cy5 positive (Figure 1A and 1B). Typically, about 2.5 mg of LMPs were obtained following this protocol. Bronchial tissue explants from C57BL/6 mice were subjected to vehicle and LMPs treatment. Histopathological analysis of bronchial sections revealed the effect of LMPs on the structural integrity of the b...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K Views.  University of Montréal. Cell membrane–shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

Video: Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Developmental Biology

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Biology

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Medicine

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...