Name: in vivo assessment of alveolar macrophage efferocytosis following ozone exposure
Uploaded: 2019-10-22T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure at JoVE.com

Denne undersøgelse er finansieret af Health Effects Institut Walter A. Rosenblith Award og NIEHS R01ES028829 (til K. M. G). Vi vil gerne takke Dr. Dianne Walters (Institut for fysiologi, ECU) for hendes hjælp til at opnå repræsentative billeder af alveolære makrofager.

Alle metoder er blevet godkendt af Udvalget for institutionel dyrepasning og-anvendelse (IACUC) i East Carolina University.
1. ozon (O3) og filtreret luft eksponering (dag 1)
<ol><li>Der må højst være 12 kvindelige C57BL/6J-mus, 8-12 uger gamle, i et stålbur (med 12 separate rum) med trådnetlåg i et O3 -eksponerings kammer.</li>
	<li>Placer termometret i eksponerings kammeret med buret for nøjagtigt at registrere temperatur og fugtighed.</li>
	<li>Tænd for den i...

<ol>
	<li>Puttur, F., Gregory, L. G., Lloyd, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+macrophages+as+the+guardians+of+tissue+repair+in+the+lung.">Airway macrophages as the guardians of tissue repair in the lung.</a> Immunology and Cell Biology. , (2019).</li><li>Gregoire, 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=Impaired+efferocytosis+and+neutrophil+extracellular+trap+clearance+by+macrophages+in+ARDS.">Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS.</a> European Respiratory Journal. 52 (2), (2018).</li><li>Fan, E. K. Y., Fan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+alveolar+macrophage+death+in+acute+lung+inflammation.">Regulation of alveolar macrophage death in acute lung inflammation.</a> Respiratory Research. 19 (1), 50 (2018).</li><li>Michlewska, S., McColl, A., Rossi, A. G., Megson, I. 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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=Diminished+airway+macrophage+expression+of+the+Axl+receptor+tyrosine+kinase+is+associated+with+defective+efferocytosis+in+asthma.">Diminished airway macrophage expression of the Axl receptor tyrosine kinase is associated with defective efferocytosis in asthma.</a> The Journal of Allergy and Clinical Immunology. 140 (4), 1144-1146 (2017).</li><li>Chen, W., Frank, M. E., Jin, W., Wahl, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+released+by+apoptotic+T+cells+contributes+to+an+immunosuppressive+milieu.">TGF-beta released by apoptotic T cells contributes to an immunosuppressive milieu.</a> Immunity. 14 (6), 715-725 (2001).</li><li>Gao, Y., Herndon, J. M., Zhang, H., Griffith, T. S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiinflammatory+effects+of+CD95+ligand+(FasL)-induced+apoptosis.">Antiinflammatory effects of CD95 ligand (FasL)-induced apoptosis.</a> Journal of Experimental Medicine. 188 (5), 887-896 (1998).</li><li>O'Brien, B. A., Fieldus, W. E., Field, C. J., Finegood, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+beta-cells+is+reduced+in+neonatal+autoimmune+diabetes-prone+rats.">Clearance of apoptotic beta-cells is reduced in neonatal autoimmune diabetes-prone rats.</a> Cell Death and Differentiation. 9 (4), 457-464 (2002).</li><li>Shen, Z. X., 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=Mineralocorticoid+Receptor+Deficiency+in+Macrophages+Inhibits+Atherosclerosis+by+Affecting+Foam+Cell+Formation+and+Efferocytosis.">Mineralocorticoid Receptor Deficiency in Macrophages Inhibits Atherosclerosis by Affecting Foam Cell Formation and Efferocytosis.</a> Journal of Biological Chemistry. 292 (3), 925-935 (2017).</li><li>Allard, B., Panariti, A., Martin, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferocytosis+as+a+regulator+of+macrophage+chemokine+receptor+expression+and+polarization.">Efferocytosis as a regulator of macrophage chemokine receptor expression and polarization.</a> European Journal of Immunology. 46 (7), 1592-1599 (2016).</li><li>Karaji, N., Sattentau, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferocytosis+of+Pathogen-Infected+Cells.">Efferocytosis of Pathogen-Infected Cells.</a> Frontiers in Immunology. 8, 1863 (2017).</li><li>Brouckaert, 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=Phagocytosis+of+necrotic+cells+by+macrophages+is+phosphatidylserine+dependent+and+does+not+induce+inflammatory+cytokine+production.">Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production.</a> Molecular Biology of the Cell. 15 (3), 1089-1100 (2004).</li><li>Gonzalez-Guevara, E., 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=Exposure+to+ozone+induces+a+systemic+inflammatory+response:+possible+source+of+the+neurological+alterations+induced+by+this+gas.">Exposure to ozone induces a systemic inflammatory response: possible source of the neurological alterations induced by this gas.</a> Inhalation Toxicology. 26 (8), 485-491 (2014).</li><li>Robertson, 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=CD36+mediates+endothelial+dysfunction+downstream+of+circulating+factors+induced+by+O3+exposure.">CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure.</a> Toxicological Sciences. 134 (2), 304-311 (2013).</li><li>Kilburg-Basnyat, B., 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=Specialized+Pro-Resolving+Lipid+Mediators+Regulate+Ozone-Induced+Pulmonary+and+Systemic+Inflammation.">Specialized Pro-Resolving Lipid Mediators Regulate Ozone-Induced Pulmonary and Systemic Inflammation.</a> Toxicological Sciences. 163 (2), 466-477 (2018).</li><li>Jakab, G. J., Spannhake, E. W., Canning, B. J., Kleeberger, S. R., Gilmour, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+ozone+on+immune+function.">The effects of ozone on immune function.</a> Environ Health Perspect. 103 Suppl 2, 77-89 (1995).</li><li>Gilmour, M. I., Hmieleski, R. R., Stafford, E. A., Jakab, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+and+recovery+of+the+alveolar+macrophage+phagocytic+system+during+continuous+exposure+to+0.5+ppm+ozone.">Suppression and recovery of the alveolar macrophage phagocytic system during continuous exposure to 0.5 ppm ozone.</a> Experimental Lung Research. 17 (3), 547-558 (1991).</li><li>Nayak, D. 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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=The+lung+environment+controls+alveolar+macrophage+metabolism+and+responsiveness+in+type+2+inflammation.">The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.</a> Nature Immunology. , (2019).</li><li>Crowther, J. E., 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=Pulmonary+surfactant+protein+a+inhibits+macrophage+reactive+oxygen+intermediate+production+in+response+to+stimuli+by+reducing+NADPH+oxidase+activity.">Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity.</a> Journal of Immunology. 172 (11), 6866-6874 (2004).</li><li>Silveyra, P., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variant+associations+of+human+SP-A+and+SP-D+with+acute+and+chronic+lung+injury.">Genetic variant associations of human SP-A and SP-D with acute and chronic lung injury.</a> Frontiers in Bioscience. 17, 407-429 (2012).</li><li>Schagat, T. L., Wofford, J. A., Wright, J. 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Efferocytose er en antiinflammatorisk proces, hvor makrofager klar apoptotiske celler og snavs samt producere flere anti-inflammatoriske mediatorer9,10,11,12,16 ,18. Flere modeller af efferocytosis har givet indsigt i, hvordan makrofag er en kritisk celle i opløsningen af inflammation6,<su...

Lungerne er konstant udsat for miljømæssige fornærmelser, herunder luft partikler, vira, bakterier, og oxidant gasser, der udløser lungebetændelse1,2,3. Disse fornærmelser kan kompromittere gasudveksling og fremkalde irreversibel vævsskade4,5. Alveolære makrofager, som udgør ca. 95% af de immunceller, der findes i murine og humane lunger ved homøostase, er kritiske regulatorer af lungebetændelse efter miljømæssige fornærmelser1,2, 3,4,5. Alveolære makrofager er afgørende i værts forsvaret ved fagocyterende og eliminere patogener. For nylig har alveolære makrofager vist sig at fremme vævs homøostase og opløsningen af inflammation gennem efferocytose6,7. Efferocytose er en Fagocytisk proces, hvor makrofager opsluge og eliminere apoptotiske celler8,9,10. Efferocytose resulterer også i produktionen af mediatorer (dvs. Il-10, TGF-β, PGE2, og nitrogenoxid), der yderligere forstærker processen, hvilket resulterer i opløsningen af inflammation9,10,11 ,12,16,18. Denne proces er nødvendig for at forebygge sekundær nekrose og fremme vævs homøostase12,13,14. Flere undersøgelser har forbundet nedsat efferocytose med forskellige kroniske lungesygdomme, herunder astma, kronisk obstruktiv lungesygdom, og idiopatisk pulmonal fibrose8,9,15, 16,17.
O3 er et kriterium luftforurenende stof, der forværrer og øger forekomsten af kroniske lungesygdomme19,20,21. O3 inducerer lungebetændelse og-skade og vides at forringe alveolær makrofag fagocytose af bakterielle patogener22,23. Det er imidlertid ukendt, om O3 hæmmer alveolær makrofag efferocytose. Undersøge O3-induceret ændringer i alveolær makrofag efferocytose vil give potentielle indblik i, hvordan eksponering kan føre til kronisk lungesygdom incidens og eksacerbation. Beskrevet nedenfor er en simpel metode til at evaluere alveolær makrofag efferocytose i lungerne hos hunmus efter akut O3 eksponering.
Den skitserede metode besidder flere fordele i forhold til andre efferocytose protokoller almindeligvis anvendes i marken ved at eliminere brugen af dyre fluorescerende farvestoffer, omfattende flow cytometri målinger, og ex vivo manipulation af alveolære makrofager24 ,25. Desuden, denne protokol foranstaltninger alveolær makrofag efferocytose i forbindelse med lunge mikromiljø, som kan påvirke makrofag funktion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annexin V-FITC Kit</td>
			<td>Trevigen</td>
			<td>4830-250-K&#160;</td>
			<td>The TACS&#160;Annexin V-FITC Kit allows rapid, specific, and quantitative identification of apoptosis in individual cells when using flow cytometry.</td>
		</tr>
		<tr>
			<td>BCL2 Jurkat T Cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC CRL-2899</td>
			<td>The BCL2 Jurkat cell line was derived by transfecting human Jurkat T cells with the pSFFV-neo mammalian expression vector containing the human BCL-2 ORF insert and a neomycin-resistant gene. Has been for models of measuring efferocytosis.&#160;</td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Thermofisher</td>
			<td>AMQAX1000</td>
			<td>It is a benchtop assay platform equipped with state-of-the-art optics, full autofocus, and image analysis software for rapid assessment of cells in suspension. Very easy to use.</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermofisher</td>
			<td>A78300003</td>
			<td>Provides economical thin-layer preparations from any liquid matrix, especially hypocellular fluids such as bronchoalveolar lavage fluid.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Thermofisher</td>
			<td>16140071</td>
			<td>Provides Nutrients to cultured cells for them to grow. It is standard for cell culture.&#160;</td>
		</tr>
		<tr>
			<td>Kwik-Diff&#160; Reagent 2, Eosin</td>
			<td>Thermofisher</td>
			<td>9990706</td>
			<td>Eosin staining that stains cytoplasm.</td>
		</tr>
		<tr>
			<td>Kwik-Diff Reagent 1, Fixative</td>
			<td>Thermofisher</td>
			<td>9990705</td>
			<td>Fixes cells to be stained by H&#38;E.</td>
		</tr>
		<tr>
			<td>Kwik-Diff Reagent 3, Methylene Blue</td>
			<td>Thermofisher</td>
			<td>9990707</td>
			<td>Methylene Blue staining that stains the nucleus.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma/Aldrich</td>
			<td>P0781-100ML</td>
			<td>Penicillin-Streptomycin is the most commonly used antibiotic solution for culture of mammalian cells. Additionally it is used to maintain sterile conditions during cell culture.</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement&#160;</td>
			<td>Thermofisher</td>
			<td>61870036</td>
			<td>RPMI 1640 Medium (Roswell Park Memorial Institute 1640 Medium) was originally developed to culture human leukemic cells in suspension and as a monolayer. RPMI 1640 medium has since been found suitable for a variety of mammalian cells, including HeLa, Jurkat, MCF-7, PC12, PBMC, astrocytes, and carcinomas. Helps grow Jurkat T cells fast and efficiently.</td>
		</tr>
		<tr>
			<td>Stratagene UV Stratalinker 1800 UV Crosslinker</td>
			<td>Cambridge Scientific&#160;</td>
			<td>16659</td>
			<td>The Stratalinker UV crosslinker is designed to induce apoptosis, crosslink DNA or RNA to nylon, nitrocellulose, or nylon-reinforced nitrocellulose membranes.</td>
		</tr>
		<tr>
			<td>Teledyne T400 ultraviolet light photometer&#160;</td>
			<td>Teledyne API</td>
			<td>T400</td>
			<td>The Model T400 UV Absorption analyzer uses a system based on the Beer-Lambert law for measuring low ranges of ozone in ambient air.</td>
		</tr>
		<tr>
			<td>Teledyne T703 Ozone calibrator</td>
			<td>Teledyne API</td>
			<td>T703</td>
			<td>Provides feedback control of the UV lamp intensity, assuring stable ozone output.</td>
		</tr>
	</tbody>
</table>

in vivo assessment of alveolar macrophage efferocytosis following ozone exposure

Ozon (O3) er et kriterium luftforurenende stof, der forværrer og øger forekomsten af kroniske lungesygdomme. O3 eksponering er kendt for at inducere lungebetændelse, men der vides ikke meget om, hvordan eksponeringen ændrer processer, der er vigtige for opløsningen af inflammation. Efferocytose er en afviklingsproces, hvor makrofager phagocytize apoptotiske celler. Formålet med denne protokol er at måle alveolære makrofag-efferocytose efter O3-induceret lungeskade og inflammation. Der er beskrevet flere metoder til måling af efferocytose; de fleste kræver dog ex vivo manipulationer. Beskrevet i detaljer her er en protokol til at måle in vivo alveolær makrofag efferocytose 24 h efter O3 eksponering, som undgår ex vivo manipulation af makrofager og fungerer som en simpel teknik, der kan bruges til præcist at repræsentere perturbationer i denne afviklingsproces. Protokollen er en teknisk ikke-intensiv og relativt billig metode, der involverer hele kroppen O3 indånding efterfulgt af orofaryngeal aspiration af apoptotiske celler (dvs., Jurkat T celler) under generel anæstesi. Alveolær makrofag efferocytose måles derefter ved let mikroskopi evaluering af makrofager indsamlet fra bronalveolær (BAL) skylning. Efferocytose måles endelig ved at beregne et efferocytisk indeks. Tilsammen kvantificerer de skitserede metoder efferocytisk aktivitet i lungerne in vivo og tjener også til at analysere de negative sundhedsmæssige virkninger af O3 eller andre inhalerede fornærmelser.

O3 eksponering er kendt for at inducere lungebetændelse og-skade, og efferocytose er nødvendig for at opretholde vævs homøostase. C57BL/6J hunmus blev udsat for filtreret luft (FA) eller 1 ppm O3 for 3 h og nekropsied 24 h efter eksponering for at undersøge lungebetændelse og tilskadekomst. O3-udsatte mus viste en signifikant stigning i makrofager og neutrofiler i luftrummet sammenlignet med FA Control Group (figur 1<str...

Watch this Scientific Journal Video about In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure at JoVE.com

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Dette manuskript beskriver en protokol til bestemmelse af, hvorvidt udsættelse for ozon, et kriterium luftforurenende stof, hæmmer alveolær makrofag efferocytose in vivo. Denne protokol udnytter almindeligt anvendte reagenser og teknikker og kan tilpasses til flere modeller af lungeskade til at bestemme virkninger på alveolær makrofag efferocytose.

In vivo-vurdering af alveolær makrofag-Efferocytose efter ozoneksponering

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

Den protokol, der er beskrevet her, måler lungemakrofage efferocytose efter eksponering for et kriterium luftforurenende ozon. Disse data viser, at ozoneksponeringen nedsætter alveolær makrofag efferocytose og dermed kan være en mekanisme til, hvorfor forhøjede niveauer af ozon øger forekomsten af lungesygdomme. Den største fordel ved denne teknik er, at det giver mulighed for en in vivo vurdering af alveolær makrofag efferocytose uden ex vivo manipulationer af makrofager, der kan ændre deres fænotype eller funktion.Denne teknik har afsløret en ny mekanisme, hvorigennem lungen ikke kan reparere sig selv efter udsættelse for luftforurening. Derudover kan denne teknik afsløre nye veje til mål, der ændrer efferocytose i lungerne. Denne metode kan anvendes på enhver model af lungeskade eller betændelse.Men, brugeren bliver nødt til at optimere, når det er bedst at vurdere efferocytose efter lungeskade. Udførelse af oropharyngeal aspiration kan være teknisk udfordrende. Jeg vil råde arbejde med din veterinære personale for at sikre, at du har optimeret din teknik til at udføre denne procedure.Begynd med at dyrke Jurkat T-celler i basalcellekulturmedium suppleret i henhold til manuskriptretningerne ved 37 grader Celsius og 5 % kuldioxid. Jurkat T-celler er en suspension cellelinje, der kan opretholdes gennem passerer i forvarmte dyrkning medier hver tredje dag. Forbered apoptotiske celler ved at dyrke dem til 90% sammenløbet, som tager tre til fire dage efter passaging.Celler dyrkes i fem T75-kolber for at opnå et tilstrækkeligt antal celler til denne protokol. Når cellerne er klar, aspirere kolbens indhold ca. 24 milliliter og overføres til et konisk rør på 50 milliliter. Tæl cellerne ved hjælp af Trypan Blue farvning og et hæmocytometer.Cellerne pellets ved centrifugering ved 271 gange g i fem minutter, og supernatanten kasseres. Derefter resuspenderede cellerne i medierne for at opnå tre millioner celler pr. milliliter. Aliquot cellerne i 100 af 20 millimeter væv kultur retter ved at overføre fem milliliter celler pr fad.Forbered ca. ni kulturretter. Sæt en skål til side som en ueksponeret kontrol og udsætte de resterende retter til UV. Indstil UV crosslinker til det korrekte energiniveau ved at trykke på energiknappen og indtaste 600 med nummerblokken. Bestråling alle retter med cellerne med undtagelse af kontrollen og sørg for at fjerne det øverste dæksel af vævskulturskålen under UV-eksponering.Derefter inkuberes alle retterne i en cellekulturinkubator ved 37 grader Celsius og 5% kuldioxid i fire timer. Efter inkubationen kombineres alle de bestrålede celler i et konisk rør på 50 milliliter, og de pelleteres ved centrifugering ved 271 gange g i fem minutter. Kassér supernatanten og opsplit cellerne i 24 milliliter steril PBS.Centrifuge røret igen og fjern supernatanten. Forbered cellerne til at gøre mus ved at opsbruge dem i PBS ved den rette koncentration. Når musen er ordentligt bedøvet, placere den i en semi-liggende liggende position.Brug kirurgisk streng bundet mellem pløkker på en skrå akryl ark bord til at suspendere musen ved maxillary fortænder. Brug et par stumpe ikke-ridged kraftskiver til let grab og trække musen tungen og indgyde apoptotiske celler i mundhulen med en P200 pipette. Dosering er vellykket, når mus laver en knitrende støj et til to sekunder efter at have modtaget dosis.Med en behandsket finger skal du forsigtigt dække musens næse, indtil den inhalerer, mens tungen trækkes tilbage. Hold næsen dækket, indtil der ikke er væske synlig i mundhulen, og musen har taget to eller flere inhalationer. Fjern musen fra vaccinationsbrættet og returnere den til buret for at gøre det muligt at komme sig efter anæstesi og sørg for at placere musen på ryggen for at forhindre snavs i at blokere nares.Efter alle mus er vågnet fra anæstesi, vent 90 minutter til at tillade alveolær makrofager at opsluge tilstrømningen af apoptotiske celler. For at indsamle lavagevæske, forberede den aflivede mus i henhold til manuskriptet retninger. Skub langsomt PBS ind i lungerne, så de kan pustes op, og træk derefter det samme volumen ud igen og sørg for, at PBS ikke forlader næseborene.Saml den samlede lavage fra hver mus i et 15 milliliterrør. Centrifuge bronchoalveolar lavage eller BAL ved 610 gange g i seks minutter ved fire grader Celsius, indsamle supernatant i en 1,5 milliliter rør, og fryse det på minus 80 grader Celsius. Pellet repræsenterer cellerne fra bronchoalveolar rummet.Tilføj en milliliter ACK røde blodlegemer lysis buffer til pellet at fjerne de resterende røde blodlegemer. Vortex og lyse i et minut på is, derefter tilføje fire milliliter PBS at stoppe lysis reaktion. Centrifuge cellerne ved 610 gange g i seks minutter ved fire grader Celsius og aspirere supernatanten med en vakuumaspirator.Derefter resuspenderede cellerne i en milliliter PBS med 10% FBS og tælle cellerne på et hæmocytometer uden Trypan Blue. Centrifuge 120 mikroliter af hver prøve på dias ved 12 gange g i tre minutter ved hjælp af medium acceleration og en cytocentrifuge. Lad objektglassene tørre natten over.Den næste dag, plette dias med hæmaxylin og eosin for at beregne både efferocytic og differentialcelletal. Derefter kan du se slides under en Brightfield-indstilling på et biologisk mikroskop ved hjælp af en 20X- eller 40X-målsætning. Det efferocytiske indeks beregnes ud fra forholdet mellem alveolær makrofager, der fagocytoserede apoptotiske Jurkat T-celler, til alveolær makrofag uden apoptotisk celleoptagelse, og sørg for at tælle mindst 200 celler fra hvert dias.Denne protokol blev anvendt til at undersøge effekten af ozoneksponering på lungebetændelse. Ozon eksponerede mus viste en stigning i makrofager og neutrofiler i luftrummet i forhold til den filtrerede luftkontrolgruppe og havde en stigning i BAL protein, en markør for alveolær epiteldysfunktion. Før du doser musene, blev apoptose i Jurkat T-celler bekræftet med flowcytometri.Den 60 milli joule UV-eksponeringsniveau og fire timers inkubationstid resulterede i gentagne resultater af ca. 75% apoptotiske celler. Efferocytic makrofager blev identificeret som makrofager, der havde opslugt en Jurkat T celle sammenlignet med regelmæssige alveolær makrofager. Der var et betydeligt fald i det efferocytiske indeks for den ozonudsatte gruppe sammenlignet med de filtrerede luftkontroller, hvilket indikerer, at ozoninduceret lungebetændelse er forbundet med nedsat clearance af apoptotiske celler.Opmærksomhed på detaljer og nedskreds af eventuelle observerede forskelle er kritisk under denne procedure. Det anbefales også at blinde prøverne til analyse og har to forskellige personer tælle makrofager. Efter isolering kan BAL-cellerne anvendes til mRNA-analyse, der er farvet for yderligere markører ved immunfluorescens, og/eller køre på et flowcytometer for at skelne fænocypiciske ændringer.

Research

JoVE Journal

Immunology and Infection

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

Anvendelse af fluorescerende Skud Arrays for Vurdering af T-celle respons<em&gt; In vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

High Throughput Fluorometrisk Teknik for Vurdering af makrofag Fagocytose og actinpolymerisation

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging af Liver efter<em&gt; Leishmania</em&gt; Infektion: En Vurdering af Nedsat Hæmodynamik

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

In vivo Vurdering af gnaver Plasmodium parasitæmi og merozoit Invasion ved flowcytometri

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometri af partikelbundet Bet v 1 Allergen i PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Magnetisk omrører metode til påvisning af<em&gt; trikiner</em&gt; Larver i Muscle Prøver

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Vurdering af antistof-baserede lægemidler virkninger på Murine knoglemarven og Peritoneal makrofag aktivering

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Kvantificering af Efferocytosis af encellede Fluorescens mikroskopi

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...