Name: conjunctival commensal isolation and identification in mice
Uploaded: 2021-05-01T13:00:00
Description: Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

O financiamento do P30 DK034854 apoiou a VY, LB e estudos no Massachusetts Host-Microbiome Center e financiamento do NIH/NEI R01 EY022054 apoiado MG.

Todos os procedimentos envolvendo camundongos seguem as diretrizes do Comitê Institucional de Cuidados e Uso de Animais. Siga as diretrizes de segurança laboratorial (conforme orientado pelo departamento de Saúde e Segurança Ambiental Institucional) ao trabalhar com microrganismos e materiais potencialmente contaminados. Utilize recipientes de resíduos adequados e procedimentos de descontaminação antes do descarte de materiais contaminados potencialmente de risco biológico.
1. Preparaç?...

<ol>
	<li>Arpaia, 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=Metabolites+produced+by+commensal+bacteria+promote+peripheral+regulatory+T+cell+generation.">Metabolites produced by commensal bacteria promote peripheral regulatory T cell generation.</a> Nature. 504 (7480), 451-455 (2013).</li><li>Hooper, L. V., Littman, D. R., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+microbiota+and+the+immune+system.">Interactions between the microbiota and the immune system.</a> Science. 336 (6086), 1268-1273 (2010).</li><li>Nagpal, 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=Human-origin+probiotic+cocktail+increases+short+chain+fatty+acid+production+via+modulation+of+mice+and+human+gut+microbiome.">Human-origin probiotic cocktail increases short chain fatty acid production via modulation of mice and human gut microbiome.</a> Scientific Reports. 8 (1), 12649 (2018).</li><li>Graham, 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=Ocular+pathogen+or+commensal:+a+PCR+based+study+of+surface+bacterial+flora+in+normal+and+dry+eyes.">Ocular pathogen or commensal: a PCR based study of surface bacterial flora in normal and dry eyes.</a> Investigative Ophthalmology and Visual Science. 48 (12), 5616-5623 (2007).</li><li>Wang, 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=Sj&#246;gren-Like+Lacrimal+Keratoconjunctivitis+in+Germ-Free+Mice.">Sj&#246;gren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice.</a> International Journal of Molecular Sciences. 19 (2), 565-584 (2018).</li><li>Ham, B., Hwang, H. B., Jung, S. H., Chang, S., Kang, K. D., Kwon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+diversity+of+ocular+microbial+communities+in+diabetic+patients+compared+with+healthy+subjects.">Distribution and diversity of ocular microbial communities in diabetic patients compared with healthy subjects.</a> Current Eye Research. 43 (3), 314-324 (2018).</li><li>Doan, 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=Paucibacterial+microbiome+and+resident+DNA+virome+of+the+healthy+conjunctiva.">Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva.</a> Investigative Ophthalmology and Visual Science. 57 (13), 5116-5126 (2016).</li><li>Kugadas, A., Gadjeva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+microbiome+on+ocular+health.">Impact of microbiome on ocular health.</a> Ocular Surface. 14 (3), 342-349 (2016).</li><li>Dong, Q., 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=Diversity+of+bacteria+at+healthy+human+conjunctiva.">Diversity of bacteria at healthy human conjunctiva.</a> Investigative Ophthalmology and Visual Science. 53 (8), 5408-5413 (2011).</li><li>Zegans, M. E., Van Gelder, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+understanding+the+ocular+surface+microbiome.">Considerations in understanding the ocular surface microbiome.</a> American Journal of Opthalmology. 158 (3), 420-422 (2014).</li><li>Fleiszig, S. M., Efron, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+flora+in+eyes+of+current+and+former+contact+lens+wearers.">Microbial flora in eyes of current and former contact lens wearers.</a> Journal of Clinical Microbiology. 30 (5), 1156-1161 (1992).</li><li>Lu, 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=Neutrophil+L-Plastin+Controls+Ocular+Paucibacteriality+and+Susceptibility+to+Keratitis.">Neutrophil L-Plastin Controls Ocular Paucibacteriality and Susceptibility to Keratitis.</a> Frontiers in Immunology. 11, 547 (2020).</li><li>Johnson, T. R., Case, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Experiments in Microbiology. , (2010).</li><li>Reiner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalase+Test+Protocol.">Catalase Test Protocol.</a> American Society for Microbiology. , (2010).</li><li>UK SMI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standards for Microbiology Investigation. UK SMI. , (2014).</li><li>Sharp, S. E., Searcy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mannitol+salt+agar+and+blood+agar+plates+for+identification+and+susceptibility+testing+of+Staphylococcus+aureus+in+specimens+from+cystic+fibrosis+patients.">Comparison of mannitol salt agar and blood agar plates for identification and susceptibility testing of Staphylococcus aureus in specimens from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 44 (12), 4545-4546 (2006).</li><li>Siegman-Igra, Y., Azmon, Y., Schwartz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milleri+group+streptococcus--a+stepchild+in+the+viridans+family.">Milleri group streptococcus--a stepchild in the viridans family.</a> European Journal of Clinical Microbiology and Infectious Diseases. 31 (9), 2453-2459 (2012).</li><li>Mohan, B., Zaman, K., Anand, N., Taneja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerococcus+Viridans:+A+Rare+Pathogen+Causing+Urinary+Tract+Infection.">Aerococcus Viridans: A Rare Pathogen Causing Urinary Tract Infection.</a> Journal of Clinical and Diagnostic Research. 11 (1), 1-3 (2017).</li><li>Senneby, E., Nilson, B., Petersson, A. C., Rasmussen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+is+a+sensitive+and+specific+method+for+identification+of+aerococci.">Matrix-assisted laser desorption ionization-time of flight mass spectrometry is a sensitive and specific method for identification of aerococci.</a> Journal of Clinical Microbiology. 51 (4), 1303-1304 (2013).</li><li>Wan, S. J., 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=IL-1R+and+MyD88+contribute+to+the+absence+of+a+bacterial+microbiome+on+the+healthy+murine+cornea.">IL-1R and MyD88 contribute to the absence of a bacterial microbiome on the healthy murine cornea.</a> Frontiers in Microbiology. 9, 1117 (2018).</li><li>Ozkan, J., 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=Temporal+Stability+and+Composition+of+the+Ocular+Surface+Microbiome.">Temporal Stability and Composition of the Ocular Surface Microbiome.</a> Scientific Reports. 7 (1), 9880 (2017).</li><li>Oliver, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viable+but+non-culturable+state+in+bacteria.">The viable but non-culturable state in bacteria.</a> Journal of Microbiology. 43 (1), 93-100 (2005).</li><li>Epstein, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phenomenon+of+microbial+uncultivability.">The phenomenon of microbial uncultivability.</a> Current Opinion in Microbiology. 16 (5), 636-642 (2013).</li><li>Whelan, F. J., 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=Culture-enriched+metagenomic+sequencing+enables+in-depth+profiling+of+the+cystic+fibrosis+lung+microbiota.">Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.</a> Nature Microbiology. 5 (2), 379-390 (2020).</li><li>Raymond, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-enriched+human+gut+microbiomes+reveal+core+and+accessory+resistance+genes.">Culture-enriched human gut microbiomes reveal core and accessory resistance genes.</a> Microbiome. 7, 56 (2019).</li><li>Peto, L., 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=Selective+culture+enrichment+and+sequencing+of+feces+to+enhance+detection+of+antimicrobial+resistance+genes+in+third-generation+cephalosporin+resistant+Enterobacteriaceae.">Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.</a> PLoS One. 14 (11), 0222831 (2019).</li><li>Lauer, B. A., Masters, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+effect+of+calcium+alginate+swabs+on+Neisseria+gonorrhoeae.">Toxic effect of calcium alginate swabs on Neisseria gonorrhoeae.</a> Journal of Clinical Microbiology. 26 (1), 54-56 (1988).</li><li>Dubois, D., 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=Performances+of+the+Vitek+MS+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry+system+for+rapid+identification+of+bacteria+in+routine+clinical+microbiology.">Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology.</a> Journal of Clinical Microbiology. 50 (8), 2568-2576 (2012).</li><li>Kawakita, 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=double-blind+study+of+the+safety+and+Efficacy+of+1%D-3-Hydroxybutyrate+eye+drops+for+Dry+Eye+Disease.">double-blind study of the safety and Efficacy of 1%D-3-Hydroxybutyrate eye drops for Dry Eye Disease.</a> Scientific Reports. 6, 20855 (2016).</li><li>Lee, H. S., Hattori, T., Stevenson, W., Cahuhan, S. K., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+toll-like+receptor+4+contributes+to+corneal+inflammation+in+experimental+dry+eye+disease.">Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.</a> Investigative Ophthalmology and Visual Science. 53 (9), 5632-5640 (2012).</li><li>Simmons, K. T., Xiao, Y., Pflugfelder, S. C., de Paiva, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+response+to+lipopolysaccharide+on+the+ocular+surface+in+a+murine+dry+eye+model.">Inflammatory response to lipopolysaccharide on the ocular surface in a murine dry eye model.</a> Investigative Ophthalmology and Visual Science. 57 (6), 2444-2450 (2016).</li><li>Miller, D., Ioviano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microbial+flora+on+the+ocular+surface.">The role of microbial flora on the ocular surface.</a> Current Opinion in Allergy and Immunology. 9 (5), 466-470 (2009).</li><li>Nayyar, A., Gindina, S., Barron, A., Hu, Y., Danias, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+epigenetic+changes+caused+by+commensal+microbiota+contribute+to+development+of+ocular+disease?+A+review+of+evidence.">Do epigenetic changes caused by commensal microbiota contribute to development of ocular disease? A review of evidence.</a> Human Genomics. 14 (1), 11 (2020).</li><li>Stevenson, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+disease:+an+immune-mediated+ocular+surface+disorder.">Dry eye disease: an immune-mediated ocular surface disorder.</a> Archives of Ophthalmology. 130 (1), 90-100 (2012).</li></ol>

Devido ao estado paucibacteriano da superfície ocular, muitos laboratórios têm tido dificuldade em isolar as commensals oculares7,20, resultando em baixo número de amostras com crescimento, baixa abundância e baixa diversidade8. Este método melhora significativamente as práticas de cultura padrão4,21 pela adição de uma etapa de enriquecimento, bem como um cotonete ocular ...

O objetivo deste protocolo é aumentar o isolamento específico de micróbios aeróbicos e anaeróbicos viáveis e facultativos da conjuntiva ocular para caracterizar o microbioma ocular. Estudos extensivos têm perfilado comunidades mucosas commensais nos tratos da pele, intestino, respiratório e genital e mostram que essas comunidades influenciam o desenvolvimento do sistema imunológico e a resposta1,2,3. Comunidades commensais oculares têm sido demonstradas para mudar durante certas patologias da doença, como doença do olho seco4, síndrome de Sjogren5 e diabetes6. No entanto, a capacidade de definir uma comunidade típica de superfície ocular commensal é dificultada por sua abundância relativamente baixa em comparação com os outros sítios mucosas6,7,8. Isso gera controvérsia sobre se existe um microbioma ocular residente e se ele existe, se ele difere do microbioma da pele e, consequentemente, seu efeito local sobre o desenvolvimento e resposta do sistema imunológico inato. Este protocolo pode ajudar a resolver essa questão.
Geralmente, as abordagens para definir o nicho commensal ocular baseiam-se no sequenciamento e nas técnicas baseadas na cultura4,7,9. 16 S rDNA sequenciamento e análise BRISK7 mostram uma diversidade mais ampla do que as técnicas baseadas na cultura, mas são incapazes de diferenciar entre micróbios vivos e mortos. Uma vez que a superfície ocular é hostil a muitos micróbios devido às propriedades antimicrobísis do filme lacrimal4 gerando uma grande variedade de fragmentos de DNA, abordagens baseadas em DNA detectarão esses artefatos que podem distorcer os dados para identificação de bactérias mortas como commensais residentes em vez de contaminantes. Isso resulta na identificação e caracterização aberrante do espaço ocular como sendo maior em abundância de micróbios e diversidade10. Isso dificulta a definição do microbioma ocular residente através de métodos baseados em DNA. Considerando que as técnicas padrão baseadas na cultura são incapazes de detectar commensais porque a carga é muito baixa11. Nosso método melhora as práticas padrão usando um cotonete fino que pode atingir a conjuntiva, evitando assim a contaminação da pele vizinha, bem como o conceito de que organismos viáveis podem ser enriquecidos por uma breve cultura em meios densos de nutrientes com o objetivo de ressuscitar viável, mas não cultural, bem como, enriquecer para micróbios viáveis raros.
Os resultados, abundância relativa de commensais oculares por cotonete ocular, caracterizam o microbioma residente conjuntiva e são importantes para fins comparativos. Nossos dados mostram que há uma diferença entre a pele e a microbiota conjuntivival, bem como maior diversidade com aumento da idade e diferença de sexo específico em abundância. Além disso, essa abordagem tem encontrado diferenças commensais em camundongosnocautes 12. Este protocolo pode ser aplicado para descrever o microbioma ocular que pode variar devido a práticas de caging, geografia ou estado de doença, bem como os efeitos locais de metabólitos commensais e produtos sobre desenvolvimento e resposta do sistema imunológico.

<table>
	<tbody>
		<tr>
			<td>0.1 to 10 &#181;l pipet tip</td>
			<td>USA Scientific</td>
			<td>1110-300</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>0.5 to 10 &#181;l Eppendorf pipet</td>
			<td>Fisher Scientific</td>
			<td>13-690-026</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Fisher Scientific</td>
			<td>BD309623</td>
			<td>1 syringe for each eye swab group</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>1000 &#181; ml pipet tip</td>
			<td>USA Scientific</td>
			<td>1111-2021</td>
			<td>autoclave before use</td>
		</tr>
		<tr>
			<td>200 to 1000&#181;l Gilson pipetman (P1000)</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>1 needle per eye swab group</td>
		</tr>
		<tr>
			<td>3 % Hydrogen Peroxide</td>
			<td>Fisher Scientific</td>
			<td>S25359</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176; C Incubator</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 % Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>PX1840-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ana-Sed Injection (Xylazine 100 mg/ml)</td>
			<td>Santa Cruz Animal Health</td>
			<td>SC-362949Rx</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BBL Gram Stain kit</td>
			<td>Fisher Scientific</td>
			<td>B12539</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean paper towels</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Batting/Sterile rolled cotton</td>
			<td>CVS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable 1 ml Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-711-9AM</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>E.coli</td>
			<td>ATTC</td>
			<td>ATCC 8739</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for Gram stain and catalase tests</td>
		</tr>
		<tr>
			<td>Ketamine (100mg/ml)</td>
			<td>Henry Schein</td>
			<td>9950001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mac Conkey Agar Plates</td>
			<td>Fisher Scientific</td>
			<td>4321270</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Carolina Biological Supply</td>
			<td>784641</td>
			<td>Prepare plates according to mfr&#39;s instructions, store at 4 &#176;C for 1 week</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Labs</td>
			<td>C57/BL6J</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
			<td>for Mannitol Salt agar plates</td>
		</tr>
		<tr>
			<td>RPI Brain Heart Infusion Media</td>
			<td>Fisher Scientific</td>
			<td>50-488525</td>
			<td>prepare according to directions and autoclave</td>
		</tr>
		<tr>
			<td>SteriFlip (0.22 &#181;m pore size polyester sulfone)</td>
			<td>EMD/Millipore, Fisher Scientifc</td>
			<td>SCGP00525</td>
			<td>to sterilize anesthesia</td>
		</tr>
		<tr>
			<td>Sterile Corning Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>anesthesia preparation</td>
		</tr>
		<tr>
			<td>Sterile mouse cage</td>
			<td>Lab equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth picks (round bamboo)</td>
			<td>Kitchen Essentials</td>
			<td></td>
			<td>autoclave before use and swab preparation</td>
		</tr>
		<tr>
			<td>Trypticase Soy Agar II with 5% Sheep&#39;s Blood Plates</td>
			<td>Fisher Scientific</td>
			<td>4321261</td>
			<td>store at 4 &#176;C until ready to use</td>
		</tr>
		<tr>
			<td>Vitek target slide</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS</td>
			<td>BioMerieux Inc. Durham,NC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vitek-MS CHCA matrix solution</td>
			<td>BioMerieux Inc. Durham, NC</td>
			<td>411071</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use eye drops</td>
			<td>CVS Pharmacy</td>
			<td></td>
			<td>Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each</td>
		</tr>
	</tbody>
</table>

conjunctival commensal isolation and identification in mice

A superfície ocular já foi considerada imuno-privilegiada e abiótica, mas recentemente parece que há uma pequena, mas persistente presença commensal. A identificação e o monitoramento de espécies bacterianas na mucosa ocular têm sido desafiadores devido à sua baixa abundância e disponibilidade limitada de metodologia adequada para crescimento e identificação commensais. Existem duas abordagens padrão: métodos de sequenciamento de cultura ou sequenciamento de DNA. O primeiro método é problemático devido às bactérias recuperáveis limitadas e a segunda abordagem identifica bactérias vivas e mortas levando a uma representação aberrante do espaço ocular. Desenvolvemos um método robusto e sensível para o isolamento bacteriano, baseando-se em técnicas de cultivo microbiológico padrão. Trata-se de uma técnica baseada em swab, utilizando um cotonete fino "em laboratório" que visa a conjuntiva inferior, seguido por um passo de amplificação para genera aeróbico aeróbico e facultativo. Este protocolo nos permitiu isolar e identificar espécies conjuntivistas como Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp. A abordagem é adequada para definir a diversidade commensal em camundongos em diferentes condições de doença.

Os resultados representativos para uma placa de cotonete dos olhos que demonstram diferentes métodos de chapeamento são retratados na Figura 3A mostrando isolados morfologicamente diversos do mouse C57BL/6. Para cada isolado distinto, as colônias foram contadas na faixa e a abundância relativa, unidades de formação de colônias únicas (UFC) por cotonete de olhos, calculadas e plotadas para fins de comparação. Para caracterização microbiológica, as bactérias foram colhidas a part...

Watch this Scientific Journal Video about Conjunctival Commensal Isolation and Identification in Mice at JoVE.com

Conjunctival Commensal Isolation and Identification in Mice

Apresentado aqui é um protocolo para o isolamento e amplificação de bactérias comensais anaeróbicas aeróbicas e facultativas de camundongos anastrérmicos utilizando um cotonete de olho único e passo de enriquecimento baseado em cultura com identificação subsequente por métodos à base microbiológica e espectrometria de massa MALDI-TOF.

Isolamento e Identificação Conjuntivival Commensal em Camundongos

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Este método identifica microrganismos conjuntivistas oculares viáveis, o que é desafiador devido ao ambiente hostil dos olhos. Ele pode ajudar a responder se existe um microbioma ocular e como a presença bacteriana muda na doença ocular. Em contraste com o sequenciamento de DNA, que identifica microrganismos viáveis e não viáveis, este método identifica apenas microrganismos viáveis, resultando em uma compreensão mais clara da comunidade commensal ocular.Estudos sugerem que doenças oculares difíceis de diagnosticar, como olho seco não autoimune e autoimune, têm presença bacteriana ocular única. Este método poderia ser usado para diagnosticar doenças oculares com assinaturas microbianas distintas. Comece autoclavando a quantidade apropriada de rebatidas de algodão e palitos de dente para o número de ratos a serem varridos, em seguida, beliscar 1/2 um centímetro de comprimento pedaço de algodão rebatendo e provocá-lo puxando-o para fora puxando as bordas para formar uma camada porosa plana parando pouco antes do rebatedor desmoronar.Gire o rebatedor em torno de uma das extremidades afiadas do palito segurando levemente a peça esticada na ponta do palito enquanto é torcida. O cotonete de olho acabado terá uma camada muito fina de algodão estendida sobre a ponta que se estende aproximadamente 1/2 a um centímetro de distância da ponta. Insira os cotonetes em um pequeno béquer, cotonete de lado para baixo, em seguida, cubra e autoclave-os.Limpe a área de trabalho com um desinfetante para minimizar a contaminação e alíquota de 0,5 mililitros de infusão cardíaca cerebral estéril, ou mídia BHI em tubos de micro centrífugas estéreis de 1,5 mililitros. Tampe os tubos em um rack e coloque o rack no gelo. Configure o fluxo de trabalho da esquerda para a direita começando com a estação de anestesia do rato, que contém a gaiola com os ratos experimentais, gaiola estéril vazia, anestesia de temperatura ambiente, agulha de calibre 25 e uma seringa de um mililitro.Em seguida, prepare uma estação de limpeza ocular que contém o BHI aliquoted no gelo, cotonetes oculares esterilizados, colírios lubrificantes, recipiente de risco biológico, toalhas de papel limpas e spray de isopropanol de 70%. Por fim, configure a estação de revestimento com placas de ágar de sangue de temperatura ambiente, uma pipeta de 10 microliter, 10 microliter estérilsa de pontas descartáveis estéreis e um recipiente de resíduos de risco biológico. Certifique-se de que o mouse está devidamente anestesiado apertando uma almofada traseira.Só prossiga se não houver movimento. Atribua uma mão para manusear ratos anestesiados e as outras mãos para manusear o cotonete dos olhos e a cultura. Retire o rato da gaiola e coloque-o em cima da superfície de trabalho posicionada de lado com o olho esquerdo exposto.Pulverize as mãos com isopropanol e seque-as com uma toalha de papel. Descap o tubo de micro centrífugas BHI rotulado com a mão de manuseio de mídia dedicada e coloque os dois de volta no rack. Mergulhe a ponta revestida de algodão do cotonete do olho no BHI, em seguida, retire o cotonete do tubo enquanto gira a ponta duas vezes contra o tubo interno para remover o excesso de líquido e removê-lo.Com a mão manuseando o mouse, segure suavemente o mouse pelo escroto do pescoço. Por outro lado, coloque a ponta do cotonete dos olhos contra a região de conjunturação medial do olho esquerdo. Deprimir levemente o globo ocular e mover o cotonete em um movimento de lavagem da janela entre a pálpebra inferior e o olho 10 vezes, mantendo a pressão constante.Sem tocar na pele, remova suavemente a ponta do cotonete perpendicular para onde foi inserido. Coloque o lado do algodão do cotonete para baixo, diretamente em um tubo de micro centrífuga rotulado com mídia BHI. Aplique um colírio no olho esfregado.Se desejar, adquira cotonetes de pele ou pele para amostras de controle, esterilizando luvas adequadamente entre cada cotonete. Quando terminar, devolva o rato para a gaiola. Deixe o cotonete ficar por 10 a 15 minutos no gelo, depois esterilize as mãos enluvadas e remova o cotonete enquanto mistura a ponta na mídia para 10 rotações.Retire o cotonete girando a ponta contra a parede interna do tubo para cinco rotações e descarte-o em um recipiente de risco biológico. Repita o processo para cada mouse. Enriqueça a amostra incubando o tubo estaticamente por uma hora a 37 graus Celsius.Durante a incubação, rotule uma placa TSA de temperatura ambiente por cotonete de olho de rato ou cotonete de controle e divida-a ao meio. Remova as amostras enriquecidas da incubadora e coloque-as no gelo. Brevemente o vórtice das amostras para misturar, em seguida, aliquot 10 microliters da amostra na placa TSA e inclinar a placa para formar uma tira.Repita isso duas vezes. Do outro lado da linha divisória das placas, crie 10 pontos com 10 microliters de amostra cada. Incubar as placas a 37 graus Celsius durante 18 horas, dois dias e quatro dias em uma câmara limpa que impede a secagem das placas de ágar.Conte as colônias nas tiras. Note morfologia e calcule unidades de formação de colônias por cotonete para isolados morfologicamente semelhantes. Olhe para os pontos onde organismos únicos não são capturados nas tiras.Para facilitar a visualização, banhamos bactérias densamente nas placas média e direita. Normalmente, observamos muito menos bactérias em placas de cotonetes oculares. Note que, de vez em quando, os cotonetes oculares podem não produzir bactérias recuperáveis.Uma placa de cotonete de olho representativa com isolados morfologicamente diversos de um rato c57 preto seis é mostrado aqui. Para cada isolado distinto, as colônias foram contadas na faixa e a abundância relativa foi calculada e plotada. Para caracterização microbiológica, as bactérias foram colhidas a partir de placas individuais de cotonetes de olhos de rato para produzir uma placa TSA mestre.Quando o crescimento apareceu, foram realizados testes adicionais para caracterizar ou identificar os micróbios. A placa principal foi usada para fornecer inóculo suficiente para expandir os respectivos isolados. Para identificar isolados, uma placa TSA foi listrada e incubada durante a noite, e MALDI-TOF MS foi realizada.Exemplos de isolados aqui mostrados foram identificados como Streptococcus acidominimus e Aerococcus viridans. Níveis significativamente diferentes de organismos commensais foram recuperados de seis camundongos pretos C57 masculinos e femininos. Streptococcus acidominimus, Aerococcus viridans coagulates negativos Staphylococci e E.coli foram isolados de C57 preto seis N ratos.Ao tentar este protocolo, tenha em mente que o melhor resultado será alcançado revestindo o cotonete finamente e tomando tempo durante o passo do cotonete dos olhos. Se o acesso ao MALDI-TOF MS for limitado, os isolados poderão ser identificados por testes microbiológicos e bioquímicos.

conjunctival-commensal-isolation-and-identification-in-mice

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolamento e Análise de Cérebro-sequestrados de Leucócitos<em&gt; Plasmodium berghei</em&gt; ANKA infectadas Mice

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Imunologia e Infecção

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Rápida identificação de bactérias gram-negativas de Sangue Cultura Caldo Usando MALDI-TOF Espectrometria de Massa

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificação de sequências de localização Plasmodesmal em proteínas em Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificação e isolamento de oligopotentes e confirmada por linhagem mieloides progenitores da medula óssea de rato

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Isolamento e identificação de células do sistema imunológico Extravascular do coração

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

Um modelo de administração Oral Galleria mellonella para respostas imunes Study Commensal-Induced inata

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.