Name: alveolar macrophage phagocytosis and bacteria clearance in mice
Uploaded: 2019-03-02T14:00:00
Description: Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Dette arbeidet er støttet av grant R01HL116826 til X. Zhao.

Denne protokollen følger retningslinjene for institusjonelle Animal Care og bruk Committee (IACUC) i øst Virginia Medical School.
1. fluorescerende perler fagocytose
<ol><li>Euthanize musen (C57BL/6J, 6 ukens gamle, kvinnelige) av CO2 kvelning som IACUC protokoller for den etiske euthanasia dyr.</li>
	<li>Lå musen buk-up på en disseksjon bord dekket med papirhåndklær. PIN potene med sine lemmer spread-eagle og koble en streng under sine fortenner å trekke hodet slik at luft...

<ol>
	<li>Hussell, T., Bell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+plasticity+in+a+tissue-specific+context.">Alveolar macrophages: plasticity in a tissue-specific context.</a> Nature Reviews Immunology. 14, 81-93 (2014).</li><li>Belchamber, K. B. R., Donnelly, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Dysfunction+in+Respiratory+Disease.">Macrophage Dysfunction in Respiratory Disease.</a> Results and Problems in Cell Differentiation. 62, 299-313 (2017).</li><li>Broug-Holub, 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=Alveolar+macrophages+are+required+for+protective+pulmonary+defenses+in+murine+Klebsiella+pneumonia:+elimination+of+alveolar+macrophages+increases+neutrophil+recruitment+but+decreases+bacterial+clearance+and+survival.">Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.</a> Infection and Immunity. 65, 1139-1146 (1997).</li><li>Knapp, 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=Alveolar+macrophages+have+a+protective+antiinflammatory+role+during+murine+pneumococcal+pneumonia.">Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia.</a> American Journal of Respiratory and Critical Care Medicine. 167, 171-179 (2003).</li><li>Bhatia, M., Zemans, R. L., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chemokines+in+the+pathogenesis+of+acute+lung+injury.">Role of chemokines in the pathogenesis of acute lung injury.</a> American Journal of Respiratory Cell and Molecular Biology. 46, 566-572 (2012).</li><li>Marriott, H. 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=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infection and Immunity. 80, 1140-1149 (2012).</li><li>Greenlee-Wacker, 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=Clearance+of+apoptotic+neutrophils+and+resolution+of+inflammation.">Clearance of apoptotic neutrophils and resolution of inflammation.</a> Immunological Reviews. 273, 357-370 (2016).</li><li>Haslett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte+apoptosis+and+its+role+in+the+resolution+and+control+of+lung+inflammation.">Granulocyte apoptosis and its role in the resolution and control of lung inflammation.</a> American Journal of Respiratory and Critical Care Medicine. 160, 5-11 (1999).</li><li>Cox, G., Crossley, J., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+engulfment+of+apoptotic+neutrophils+contributes+to+the+resolution+of+acute+pulmonary+inflammation+in+vivo.">Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo.</a> American Journal of Respiratory Cell and Molecular Biology. 12, 232-237 (1995).</li><li>Groves, E., Dart, A. E., Covarelli, V., Caron, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+phagocytic+uptake+in+mammalian+cells.">Molecular mechanisms of phagocytic uptake in mammalian cells.</a> Cellular and Molecular Life Sciences. 65, 1957-1976 (2008).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Opsonic+Phagocytosis+via+Fc&#947;+Receptors+and+complement+receptors+on+macrophages.">Measuring Opsonic Phagocytosis via Fc&#947; Receptors and complement receptors on macrophages.</a> Current Protocols in Immunology. , (2011).</li><li>He, J. Q., Wiesmann, C., van Lookeren Campagne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+of+macrophage+complement+receptor+CRIg+in+immune+clearance+and+inflammation.">A role of macrophage complement receptor CRIg in immune clearance and inflammation.</a> Molecular Immunology. 45, 4041-4047 (2008).</li><li>Nagre, 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=Inhibition+of+Macrophage+Complement+Receptor+CRIg+by+TRIM72+Polarizes+Innate+Immunity+of+the+Lung.">Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.</a> American Journal of Respiratory Cell and Molecular Biology. 58 (6), 756-766 (2018).</li><li>Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+to+Determine+the+Engulfment+of+Apoptotic+Cells+by+Macrophages+using+pHrodo+Succinimidyl+Ester.">A Novel Method to Determine the Engulfment of Apoptotic Cells by Macrophages using pHrodo Succinimidyl Ester.</a> Journal of Immunological Methods. 342 (1-2), 71-77 (2009).</li><li>Su, H., Chen, H., Jen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+exercise+enhances+phagocytosis+by+murine+bronchoalveolar+macrophages.">Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages.</a> Journal of Leukocyte Biology. 69, 75-80 (2001).</li><li>Amiel, E., Lovewell, R. R., O'Toole, G. A., Hogan, D. A., Berwin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa.+evasion+of+phagocytosis+is+mediated+by+loss+of+swimming+motility+and+is+independent+of+flagellum+expression.">Pseudomonas aeruginosa. evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression.</a> Infection and Immunity. 78, 2937-2945 (2010).</li><li>Giannoni, E., Sawa, T., Allen, L., Wiener-Kronish, J., Hawgood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+Proteins+A+and+D+Enhance+Pulmonary+Clearance+of+Pseudomonas+aeruginosa.">Surfactant Proteins A and D Enhance Pulmonary Clearance of Pseudomonas aeruginosa.</a> American Journal of Respiratory Cell and Molecular Biology. 34, 704-710 (2006).</li></ol>

Når du utfører en gass exchange funksjon, konfronterer lungene vedvarende fremmede partikler, patogener og allergener. AMs gir den første linjen i forsvaret i kraft av deres viktigste funksjon, nemlig fagocytose. AMs også koordinere med andre immunceller i ødelegge patogener og oppløsningen av betennelse. Her beskrevet vi metoder for å vurdere spesielt fagocytose av AMs isolert fra mus lungene. Protokollen presentert i dette manuskriptet forklarer en detaljert studie av fagocytose både i vivo og in vitro, som ogs...

AMs er de store bosatt phagocytes i alveoli hvile scenen og en av de store aktørene av medfødte immunreaksjoner gjennom erkjennelsen og internalization inhalert patogener og fremmede partikler1,2. Det har blitt rapportert at AMs er avgjørende for den raske klarering av mange lunge patogener som P. aeruginosa og Klebsiella lungebetennelse3,4, så en mangel på AM fagocytose ofte resulterer i luftveiene infeksjoner, som akutt lungebetennelse, som forårsake høyere dødelighet og sykelighet priser.
AMs også starte medfødte inflammatorisk svar i lungene ved å produsere cytokiner og chemokines som TNF-α og IL-1β, hvilke crosstalk med andre celler av alveolar miljøet å produsere chemokines og rekruttere inflammatorisk nøytrofile, monocytter, og adaptive immunceller i lungene5. For eksempel, hjelper IL-1β produsert av AMs Prime utgivelsen av nøytrofile chemokine CXCL8 fra epitelceller6. Videre bidra AMs til fagocytose av apoptotisk polymorfonukleære leukocytter (PMNs), feil som fører til vedvarende lekkasje av intracellulær enzymer fra PMNs til omkringliggende vev, som resulterer i skade på vev og langvarig betennelse 7 , 8 , 9.
Fagocytose av AMs er formidlet av en direkte anerkjennelse av patogen-forbundet molekylær mønster på patogen overflaten av mønster anerkjennelse receptors (PRRs) av AMs eller ved binding av opsonized patogener med immun effektor reseptorer av AMs 10. for sistnevnte AMs kan gjenkjenne mål opsonized med immunglobulin (IgG) gjennom deres Fcγ reseptorer (FcγR) eller patogener belagt med supplement fragmenter, C3b og C3bi, gjennom deres supplement reseptorer (CR)11. Blant supplement reseptorer, CR av immunglobulin gruppe (CRIg) er uttrykt selektivt i vev makrofager12, og en siste finne valgte CRIg i AM fagocytose i sammenheng med P. aeruginosa lungebetennelse 13.
Mange opprinnelige studier bruk metoder for å vurdere macrophage fagocytose å beskrive molekylære mekanismer macrophage funksjon14,15. Men krever metodene som i vivo fagocytose en presis kvantifisering av fagocytose. Her vi oppsummere en detaljert metodikk for både i vitro og in vivo fagocytose bruker fluorescein isothiocyanate (FITC)-glass perler og P. aeruginosa grønne fluorescerende protein (GFP), henholdsvis. Videre forklare vi metoden skille blant PRR - CR- og FcγR-mediert fagocytose. Til slutt, vi rapportere en metode for å beskrive bakteriell klaring musen med hensyn til P. aeruginosa lungebetennelse.

<table border="0" cellpadding="0" cellspacing="0" style="width:809px;" width="809">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18-G Needle</td>
			<td style="width:179px;">Nipro Medical&#160;</td>
			<td style="width:143px;">CI+1832-2C</td>
			<td style="width:272px;">Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,7-diaminofluorene (DAF)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">D17106</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:179px;">Decon Labs Inc.</td>
			<td style="width:143px;">18C27B</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plate</td>
			<td style="width:179px;">Corning</td>
			<td style="width:143px;">3603</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK lysis buffer</td>
			<td style="width:179px;">Life Technologies</td>
			<td style="width:143px;">A10492</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa fluor-488 Zymosan-A-bioparticle</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">Z23373</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C5 deficient serum&#160;</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">C1163</td>
			<td>Biochemical reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:179px;">Labnet International</td>
			<td style="width:143px;">C0160-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytospin 4 Cytocentrifuge</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">A78300101 Issue 11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Cell Culture Media</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">11995-065</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:179px;">Atlanta Biologicals</td>
			<td style="width:143px;">S11550</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:143px;">FACSCalibur</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow Jo Software</td>
			<td style="width:179px;">FlowJo, LLC</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:179px;">Dumont</td>
			<td style="width:143px;">0508-SS/45-PS-1</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-carboxylated latex beads</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">L4530</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-P. aeruginosa</td>
			<td style="width:179px;">ATCC</td>
			<td>101045GFP</td>
			<td>Suitable for&#160; cell infection assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass bottom dish</td>
			<td style="width:179px;">MatTek Corp.</td>
			<td>P35G-0.170-14-C</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-Pressure Syringe</td>
			<td style="width:179px;">Penn-Century</td>
			<td>FMJ-250</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Homogenizer</td>
			<td style="width:179px;">Omni International</td>
			<td>TH-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">H1009</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted Fluorescence Microscope</td>
			<td style="width:179px;">Olympus</td>
			<td style="width:143px;">IX73</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride</td>
			<td style="width:179px;">Hospira</td>
			<td style="width:143px;">CA-2904</td>
			<td>Pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shandon Kwik-Diff Stains</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">9990700</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Agar</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1425</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1427</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MicroSprayer Aerosolizer</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">IA-1C</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6148</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">20012-027</td>
			<td>Cell Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-SRBC-IgG&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55806</td>
			<td>Suitable for immuno-assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-SRBC-IgM&#160;</td>
			<td style="width:179px;">Cedarline Laboratories</td>
			<td style="width:143px;">CL9000-M</td>
			<td>Suitable for immuno-assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td style="width:179px;">Miltex</td>
			<td style="width:143px;">5-2</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">LS-2</td>
			<td>Suitable for laboratory animal use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:179px;">BioRad</td>
			<td style="width:143px;">1610301</td>
			<td>Analytical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Med)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">15012-12</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Small)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">91500-09</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sheep red blood cells (SRBCs)&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55876</td>
			<td>Washed, preserved SRBCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">U5378</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine&#160;</td>
			<td style="width:179px;">Akorn Animal Health</td>
			<td style="width:143px;">59399-110-20</td>
			<td>Pharmaceutical grade</td>
		</tr>
	</tbody>
</table>

alveolar macrophage phagocytosis and bacteria clearance in mice

Alveolar makrofager (AMs) vakt alveolar løpet av lungene. Fagocytose av AMs spiller en avgjørende rolle i forsvaret mot invaderende patogener, fjerning av død celler eller fremmede partikler og i oppløsning av inflammatoriske svar og vev remodeling, behandler som er formidlet av ulike overflate reseptorer av AMs. Her rapporterer vi metoder for analyse av den phagocytic AMs bruker i vitro og in vivo analyser og eksperimentelle strategier for å skille mellom den mønster anerkjennelse reseptor - supplement reseptor- og Fc gamma reseptor-mediert fagocytose. Til slutt, vi diskuterer en metode for å etablere og karakterisere P. aeruginosa lungebetennelse modell i mus å vurdere bakteriell klarering i vivo. Disse analyser representerer de vanligste metodene for å evaluere AM funksjoner og kan også brukes til å studere macrophage funksjon og bakteriell klarering i andre organer.

Vi først fremført eksperimentet å analysere fagocytose av musen primære AMs. Gjennom alle analyser sammenlignet vi AMs isolert fra WT og TRIM72KO mus. Som vist i figur 1A, avslørt fluorescens mikroskopi at fagocytose av FITC glass perler av musen primære AMs oppstår etter 1 time med inkubering. Figur 1 B viser analyse av fagocytose av flowcytometri. Kvantifisering av fagocytose ...

Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar Macrophage fagocytose og bakterier klarering i mus

Her rapporterer vi vanlige metoder for å analysere phagocytic funksjon murine alveolar makrofager og bakteriell klaring fra lungene. Disse metodene studere i vitro fagocytose av fluorescein isothiocyanate perler og i vivo fagocytose Pseudomonas aeruginosa Green fluorescerende protein. Vi har også beskriver en metode for å fjerne P. aeruginosa i mus.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Pseudomonas aeruginosa er et BSL-2 nivå patogen. Husk å følge alle sikkerhetsrutiner på BSL-2-nivå ved håndtering av denne organismen. Her vil vi demonstrere primær celleisolasjon, in vitro phagocytosis og fagocytisk veianalyse, og in vivo phagocytosis og bakteriell clearance vurdering hos mus.Vellykket intratrakeal levering krever praksis å mestre. Kontroller at mikrosprayeren er riktig lastet, nålen er mellom de to vokalfoldene, og stempelhastigheten er riktig kontrollert. Begynn med å feste musen i liggende stilling med lemmer spredt på et disseksjonsbrett dekket med papirhåndklær og hekte en streng under fortennene for å trekke tilbake hodet slik at luftrøret er plassert rett og jevnt.Desinfiser musen med 70% etanol og bruk vanlige tang for å trekke opp huden i midten av kroppen. Bruk kirurgisk saks for å kutte huden fra magen til toppen av halsen. Bruk den stumpe enden av standard kirurgisk saks for å forsiktig dissekere halsmuskelen og bindevev.Åpne bukveggen under brystkassen. Klipp membranen, og kutt bort den nedre delen av brystkassen for å delvis eksponere lungene. Bruk vårsaks til å eksponere luftrøret og bruk tangen til å gripe tak i en bruskring.Bruk mikrosaks til å forsiktig gjøre et ca. 1,5 millimeter snitt på luftrørets ventrale ansikt. Streng forsiktig en kort lengde av suturtråd under luftrøret og sett en 18 gauge kanyle inn i luftrøret. Når kanylen er på plass, forsiktig lavage lungen tre ganger med en milliliter frisk PBS per vask, forsiktig trekke væsken inn i sprøyten før du reinfusing den tilbake i lungene for tre ganger på rad.Etter hvert toalett, overføre den oppsamlede bronkoalveolar lavage væske eller BALF i en 15-milliliter konisk rør for sentrifugering av den totale høstet væske. Resuspend pellet i en milliliter fersk PBS for en andre sentrifugering og resuspend vasket alveolr makrofager i to milliliter av Dulbecco's Modified Eagles Medium eller DMEM supplert med 10% ikke-varme-inaktivert fosterbovin serum. Deretter overfører du de primære alveolære makrofagene til en petriskål med glassbunn for deres to-dagers kultur ved 37 grader Celsius i en celleinkubator.Etter 48 timer vask kulturen med en milliliter PBS før du mater kulturen med to milliliter friskt medium. Deretter legger du til 50 to-mikrometer diameter karboksylert latex FITC-konjugert perler per celle til kulturen for en times inkubasjon ved 37 grader Celsius i en celleinkubator. På slutten av inkubasjonen vasker du platen mye fem ganger med en milliliter frisk PBS per vask for å fjerne ekstracellulære perler og tilfeldig bilde 100 celler for å telle cellene som inneholder intracellulære perler.For en opsoniseringsanalyse, inkuber to ganger 10 til åttende sau røde blodlegemer eller SRBCer med 50 mikroliter kanin anti-SRBC immunoglobulin M eller IgM i 30 minutter ved romtemperatur. Inkuber deretter de opsoniserte SRBCene med 50 mikroliter C5-mangelfull humant serum i 30 minutter ved 37 grader Celsius for å fikse C3b- og C3b-hemmeren utfyller fragmenter på IgM-belagte SRBCer. Deretter aspirerer media og legger til 100 mikroliter av en ganger 10 til de syv opsoniserte SRBCene per milliliter medium til hver brønn av en 96-brønnsplate som inneholder en ganger 10 til fjerde natt-kultiverte murine makrofager per brønn.Inkuber SRBC-musemakrofagkokulturen i en time ved 37 grader Celsius, og fjern deretter de ubundne SRBCene ved å vaske med 100 mikroliter ammoniumkloridkaliumlysbuffer i ett minutt. Etter at de ubundne SRBCene er fjernet, skyll med 100 mikroliter medier. Lyse de resterende cellene med 0,1% natrium dodecylsulfat og behandle lysates med 50 mikroliter på 2, 7-diaminofluoren supplert med 3% hydrogenperoksid og seks molar urea.Mål deretter absorbansen av hemoglobinkatalysert fluorblå formasjon på et spektrofotometer ved 620 nanometer. Bruk en standardkurve ved 620 nanometer absorbansverdier med kjent antall SRBCer for å bestemme antall opsoniserte SRBCer som er fagocytisert. For å vurdere mønstergjenkjenning reseptor-mediert fagocytose, vask to-dagers kultivert mus primære alveolære makrofager med en milliliter PBS og behandle cellene med 500 mikroliter av friskt medium som inneholder 100 Alexa fluor-488 konjugert Zymosan-A-biopartikler.Etter en time ved 37 grader Celsius, arrester fagocytose med 500 mikroliter iskald PBS og vask cellene mye fem ganger med en milliliter PBS per vask. Etter siste vask, fest cellene med 4% paraformaldehyd i 10 minutter ved romtemperatur og vask cellene fem ganger som demonstrert. Etter siste vask dekker cellene med 500 mikroliter frisk PBS for avbildning under differensial interferenskontrast og fluorescerende kanal ved 488 nanometer for å kvantifisere antall Zymosan-A-biopartikkelholdige alveolære makrofager.For in vivo alveolar makrofag phagocytosis evaluering, bekrefter riktig nivå av sedasjon ved mangel på respons på tå klemme i en bedøvet mus og plassere musen på et flatt bord med en plasttråd under de øvre fortenner. Plasser musen i en semi-liggende rostrum på 45 graders posisjon med ventral overflate vendt oppover og bruk buede tang for å trekke ut og undertrykke tungen. Sett deretter inn en mikrosprayer mellom vokalfoldene for å intratracheally administrere 50 mikroliter på fem ganger 10 til de seks kolonidannende enhetene til P.aeruginosa GFP i lungene til det bedøvede dyret.For å bekrefte at mikrosprayernålen er i luftrøret, flytt sprøyten forsiktig og følg stemmefoldene på hver side av nålen. En time etter infeksjonen, samle toalettvæsken som nettopp demonstrert og pellet alveolr makrofager ved sentrifugering. Gjenoppbruk en ganger 10 til de tredje cellene i 100 mikroliter frisk PBS for cytocentrifuge på et glass lysbilde.Deretter differensialt flekker cytospunnet lysbilder for alveolære makrofager, nøytrofiler, og lymfocytter, i henhold til standard histokjemiske protokoller. Velg tilfeldig 100 alveolære makrofager for å kvantifisere prosentandelen av celler som fagocytosed bakterier. I den første in vivo bakterier clearance studien, intratracheally injisere en sublethal dose på ca 2,5 til fem ganger 10 den femte kolonidannende enheter per milliliter av P.aeruginosa i bedøvet vill type og muterte mus og måle kroppsvekten til hvert dyr hver dag i seks dager.I den andre studien injiserer du et nytt sett med villtype og muterte mus med dødelig tre ganger 10 til de syv kolonidannende enhetene per milliliter dose p.aeruginosa og registrerer dødeligheten innen to dager etter en injeksjon, samle hele lungevevet på dødstidspunktet eller to dager etter injeksjonen for kvantifisering av lungebakteriell byrde ved maksimal infeksjon. Etter høsting lungene, kutte lungeprøver i små biter på is og homogenisere lungefragmenter ved hjelp av en justert elektrisk homogenisator innstilling. Deretter plate 100 mikroliter homogenate på Pseudomonas isolasjon agar plater i 10-fold seriell fortynninger.Fluorescensmikroskopi avslører at musen primær alveolar makrofagfag phagocytosis av FITC latex perler oppstår etter en time med inkubasjon, med TRIM72 knockout makrofager som viser en betydelig høyere fagocytisk evne. Omvendt resulterer overuttrykk av TRIM72, et protein med ukjent funksjon i murinmakrofagceller, en mer enn fem ganger reduksjon i komplementfagocytose. Alexa fluor-488 konjugert Zymosan-A partikler, men inn er inndelt i like mengde av primære alveolære makrofager isolert fra enten vill type eller knockout mus.Differensialfarging av BALF høstet fra vill type og knockout dyr avslører et lignende antall makrofager, men en høyere fagocytisk evne for makrofagene høstet fra knockout mus. Videre viser TRIM72 knockout mus en raskere gjenoppretting av kroppsvekten, opprettholder overlevelsen og viser en lavere bakteriell byrde enn ville type dyr etter intratracheal P.aeruginosa administrasjon. Ved hjelp av denne teknikken kan andre fagocytiske prosesser som er viktige for lungebetennelse som nøytrofil fagocytose og det relative bidraget fra andre fagocytter i bakterieclearance analyseres.I kombinasjon med farmakologiske hemmere, adaptiv overføring og transgene dyr, kan denne teknikken hjelpe forskere til å utforske den molekylære komponenten av spesifikk type fagocytose for fagocytter av interesse.

alveolar-macrophage-phagocytosis-and-bacteria-clearance-in-mice

Research

JoVE Journal

Immunology and Infection

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Bone Marrow-avledet Macrophage Produksjon

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

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

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Høy gjennomstrømning Fluorometrisk Teknikk for Vurdering av Macrophage Fagocytose og Actin Polymerisering

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

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Makrofag Kolesterol Nedbryting og dens effekt på Fagocytose av<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Fagocytoseanalyse for apoptotiske celler i<em&gt; Drosophila</em&gt; Embryoer

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolasjon og In Vitro kultur Murine og menneskelig Alveolar makrofager

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

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Bruke forbedret grønne fluorescens Protein-uttrykke Escherichia Coli å vurdere musen Peritoneal Macrophage fagocytose

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Assaying for uorganiske Polyphosphate i bakterier

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Behandling av lunges tømming Fluid og matchet Blood for Alveolære macrophage og CD4+ T-Cell IMMUNFENOTYPING og HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

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.

I vivo vurdering av Alveolære macrophage Efferocytosis etter ozon eksponering

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

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Måle fagocytose av Aspergillus fumigatus Conidia av menneskelige leukocytter bruker Flow flowcytometri

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...