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

P30 DK034854'ten sağlanan finansman, Massachusetts Host-Microbiome Center'daki VY, LB ve çalışmaları ve NIH/NEI R01 EY022054'ten gelen fon MG'yi destekledi.

Farelerle ilgili tüm prosedürler Kurumsal Hayvan Bakım ve Kullanım Komitesi yönergelerine uyar. Mikroorganizmalar ve potansiyel olarak kontamine olmuş malzemelerle çalışırken laboratuvar güvenliği yönergelerini (Kurumsal Çevre Sağlığı ve Güvenliği departmanınız tarafından yönlendirildiği şekilde) izleyin. Biyolojik tehlikeyle kirlenmiş olabilecek malzemelerin bertaraf edilmeden önce uygun atık kaplarını ve dekontaminasyon prosedürlerini kullanın.
1. Göz çubuğu...

<ol>
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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. 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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. 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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. 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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. 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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>

Oküler yüzeyin paucibacterial durumu nedeniyle, birçok laboratuvar oküler commensals7,20izole etmektezorlandı,bu da büyüme, düşük bolluk ve düşük çeşitlilik ile düşük numune sayısına neden oldu8. Bu yöntem, bir zenginleştirme adımının yanı sıra MALDI-TOF MS tarafından yeniden tasarlanmış bir göz çubuğu ve tanımlaması eklenerek standart kültür uygulamaları4,<sup class=...

Bu protokolün amacı, oküler mikrobiyomu karakterize etmek için oküler konjonktivadan canlı ve nadir aerobik ve öğretim üyesi anaerobik mikropların spesifik izolasyonunu arttırmaktır. Kapsamlı çalışmalar cilt, bağırsak, solunum ve genital sistemlerdeki kommensal mukozal toplulukların profilini çıkarmıştır ve bu toplulukların bağışıklık sisteminin gelişimini ve yanıt1, 2,3'üetkilediğini göstermektedir. Göz eti kommenal topluluklarının Kuru göz hastalığı4, Sjogren sendromu5 ve diyabet6gibi bazı hastalık patolojileri sırasında değiştiği gösterilmiştir. Yine de, tipik bir oküler yüzey kommenal topluluğunu tanımlama yeteneği, diğer mukozal sitelere kıyasla nispeten düşük bollukları ile engellenir6,7,8. Bu, yerleşik bir oküler mikrobiyom olup olmadığı ve varsa, cilt mikrobiyomundan farklı olup olmadığı ve sonuç olarak doğuştan gelen bağışıklık sistemi gelişimi ve yanıtı üzerindeki yerel etkisi hakkında bir tartışmaya neden olur. Bu protokol bu sorunun çözülmesine yardımcı olabilir.
Genel olarak, oküler kommensal niş tanımlamak için yaklaşımlar sıralama ve kültür tabanlı tekniklere dayanır4,7,9. 16 S rDNA dizilimi ve BRISK analizi7, kültür tabanlı tekniklerden daha geniş bir çeşitlilik gösterir, ancak canlı ve ölü mikroplar arasında ayrım yapamaz. Göz yaşartıcı filmin anti-mikrobiyal özellikleri4 nedeniyle göz yüzeyi birçok mikroplara düşman olduğundan, DNA tabanlı yaklaşımlar, ölü bakterilerin kirleticiler yerine yerleşik kommensallar olarak tanımlanmasına yönelik verileri çarpıtabilecek bu eserleri tespit edecektir. Bu, oküler alanın mikrop bolluğu ve çeşitliliğinde daha yüksek olarak anormal kommensal tanımlama ve niteleme ile sonuçlanır10. Bu, yerleşik oküler mikrobiyomun DNA tabanlı yöntemlerle tanımlanmasını zorlaştırır. Standart kültür tabanlı teknikler, yük çok düşük olduğundan virgülleri algılayamaz11. Yöntemimiz, konjonktivayı hedefleyebilen ince bir çubuk kullanarak standart uygulamaları geliştirir, böylece komşu ciltlerden kontaminasyonu önler, ayrıca canlı organizmaların canlı ama kült olmayan mikropları canlandırmak ve nadir uygulanabilir mikroplar için zenginleştirmek amacıyla besin yoğun ortamlarında kısa kültürle zenginleştirilebileceği kavramını da önler.
Sonuçlar, göz çubuğu başına göreceli oküler kommensal bolluğu, konjonktiva yerleşik mikrobiyomu karakterize eder ve karşılaştırmalı amaçlar için önemlidir. Verilerimiz, cilt ve konjonktival mikrobiyota arasında bir fark olduğunu, ayrıca artan yaş ve bol miktarda cinsiyete özgü bir fark ile daha fazla çeşitlilik olduğunu göstermektedir. Ayrıca, bu yaklaşım tekrar tekrar nakavt farelerde övgüye yersiz farklılıklar buldu12. Bu protokol, kafes uygulamaları, coğrafya veya hastalık durumuna bağlı olarak değişebilen oküler mikrobiyomun yanı sıra kommensal metabolitlerin ve ürünlerin bağışıklık sistemi gelişimi ve yanıtı üzerindeki yerel etkilerini tanımlamak için uygulanabilir.

<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

Oküler yüzey bir zamanlar immün ayrıcalıklı ve abiyotik olarak kabul edildi, ancak son zamanlarda küçük, ancak kalıcı bir kommensal varlık olduğu görülüyor. Oküler mukozadaki bakteri türlerinin tanımlanması ve izlenmesi, düşük bollukları ve kommensal büyüme ve tanımlama için uygun metodolojinin sınırlı metodolojisi nedeniyle zor olmuştur. İki standart yaklaşım vardır: kültür tabanlı veya DNA dizileme yöntemleri. İlk yöntem, sınırlı geri kazanılabilir bakteriler nedeniyle sorunludur ve ikinci yaklaşım, oküler alanın anormal bir temsiline yol açan hem canlı hem de ölü bakterileri tanımlar. Standart mikrobiyolojik kültleme tekniklerini geliştirerek bakteri izolasyonu için sağlam ve hassas bir yöntem geliştirdik. Bu, alt konjonktivayı hedefleyen "laboratuvar içi" yapılmış ince bir çubuk kullanan, ardından aerobik ve öğretim üyesi anaerobik cins için bir amplifikasyon adımı kullanan çubuk tabanlı bir tekniktir. Bu protokol, Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., vb. Yaklaşım, farklı hastalık koşullarında farelerde kommensal çeşitliliği tanımlamak için uygundur.

Kaplama için farklı yöntemleri gösteren bir göz çubuğu plakası için temsili sonuçlar, C57BL / 6 fareden morfolojik olarak farklı izolatları gösteren Şekil 3A'da resmedilmiştir. Her ayrı izole için, koloniler şeritte sayıldı ve göreceli bolluk, göz çubuğu başına benzersiz Koloni Şekillendirme Birimleri (CFO'lar), karşılaştırma amacıyla hesaplandı ve çizildi. Mikrobiyolojik karakterizasyon için, bakteriler ana bir TSA plakası üretmek için tek tek fare göz...

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

Conjunctival Commensal Isolation and Identification in Mice

Burada sunulan, mikrobiyolojik tabanlı yöntemler ve MALDI-TOF kütle spektrometresi ile daha sonra tanımlanması ile benzersiz bir göz çubuğu ve kültür tabanlı zenginleştirme adımı kullanılarak aerobik ve öğretim üyesi anaerobik fare konjonktival commensal bakterilerin izolasyonu ve amplifikasyonu için bir protokoldür.

Farelerde Konjonktival Kommenal İzolasyon ve Tanımlama

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

Bu yöntem, gözlerin düşmanca ortamı nedeniyle zorlayıcı olan uygulanabilir oküler konjonktival mikroorganizmaları tanımlar. Oküler mikrobiyomun var olup olmadığını ve göz hastalığında bakteri varlığının nasıl değiştiğini cevaplana yardımcı olabilir. Hem uygulanabilir hem de canlı olmayan mikroorganizmaları tanımlayan DNA diziliminin aksine, bu yöntem sadece uygulanabilir mikroorganizmaları tanımlar ve bu da öküler kommenal topluluğun daha net anlaşılmasını sağlamakla sonuçlanır.Çalışmalar, otoimmün olmayan ve otoimmün kuru göz gibi göz hastalıklarını teşhis örülmenin zor olduğunu ve benzersiz oküler bakteri varlığına sahip olduğunu göstermektedir. Bu yöntem, ayırt edici mikrobiyal imzalara sahip göz hastalıklarını teşhis etmek için kullanılabilir. Sürünecek fare sayısı için uygun miktarda pamuk vuruşu ve kürdanı otomatik olarak kapatarak başlayın, ardından 1/2 santimetre uzunluğunda pamuk vuruşu parçasını kıstırın ve vuruş dağılmadan hemen önce duran düz bir gözenekli tabaka oluşturmak için kenarları çekerek alay edin.Bükülmüş olarak gerilmiş parçayı kürdan ucunda hafifçe tutarak vuruşu kürdanın keskin uçlarından birinin etrafında döndürün. Bitmiş göz çubuğu, uçtan yaklaşık 1/2 ila bir santimetre uzağa uzanan ucun üzerine gerilmiş çok ince bir pamuk tabakasına sahip olacaktır. Sürüntüleri küçük bir behere yerleştirin, aşağı doğru sürün, sonra örtün ve otoklavlayın.Kontaminasyonu ve 0,5 mililitre steril beyin kalbi infüzyonunu veya BHI ortamını etiketli 1,5 mililitre steril mikro santrifüj tüplerine en aza indirmek için çalışma alanını bir dezenfektanla temizleyin. Tüpleri bir rafa takın ve rafı buza yerleştirin. Deneysel fareler, boş steril kafes, oda sıcaklığı anestezisi, 25 kalibre iğne ve bir mililitre şırınga ile kafesi içeren fare anestezi istasyonundan başlayarak soldan sağa iş akışını ayarlayın.Daha sonra, buz üzerinde aliquoted BHI, sterilize göz çubukları, yağlama damlaları, biyolojik tehlike kabı, temiz kağıt havlular ve% 70 izopropanol spreyi içeren bir göz tarama istasyonu hazırlayın. Son olarak, oda sıcaklığında kan agar plakaları, 10 mikroliter pipet, 10 mikroliter steril tek kullanımlık uç ve biyolojik tehlike atık kabı ile kaplama istasyonunu kurun. Bir arka ayak pedi sıkarak farenin düzgün bir şekilde uyuşturulduğından emin olun.Sadece hareket yoksa devam edin. Anesteziye bulaşmış fareleri işlemek için bir el, göz bezini ve kültürü işlemek için diğer ellerinizi atayın. Fareyi kafesten çıkarın ve sol gözü açıktayken yan tarafına yerleştirilmiş çalışma yüzeyinin üzerine yerleştirin.Eldivenli ellere izopropanol püskürtün ve kağıt havlu ile kurulayın. Etiketli BHI mikro santrifüj tüpünü özel medya işleme eliyle çıkarın ve ikisini rafa geri yerleştirin. Göz küreğinin pamuk kaplı ucunu BHI'ye batırın, ardından fazla sıvıyı çıkarmak ve çıkarmak için ucu iç tüpe iki kez döndürürken küveti tüpten çekin.Fare taşıma eliyle, fareyi boynun scruff'ından hafifçe tutun. Diğer taraftan, göz bezinin ucunu sol gözün medial konjonktival bölgesine yerleştirin. Göz küresini hafifçe bastırın ve bezi alt göz kapağı ile göz arasındaki bir pencere yıkama hareketinde 10 kez hareket ettirerek sürekli basıncı koruyun.Kürke dokunmadan, çubuğun ucunu yerleştirildiği yere dik olarak hafifçe çıkarın. Çubuk pamuk tarafını doğrudan BHI ortamlı etiketli bir mikro santrifüj tüpüne yerleştirin. Sürünmüş göze bir göz damlası uygulayın.İsterseniz, kontrol numuneleri için cilt veya kürk sürüntüleri alın, eldivenleri her çubuk arasında uygun şekilde sterilize edin. İşiniz bittiğinde, fareyi kafese geri döndürün. Bez buz üzerinde 10 ila 15 dakika bekletin, ardından eldivenli elleri sterilize edin ve ucu 10 dönüş boyunca medyada karıştırırken çubuğu çıkarın.Ucu tüpün iç duvarına doğru beş dönüş boyunca döndürerek çubuğu çekin ve biyolojik tehlike kabına atın. Her fare için işlemi yineleyin. Tüpü statik olarak 37 santigrat derecede bir saat inkübe ederek numuneyi zenginleştirin.Kuluçka sırasında, fare gözü çubuğu başına bir oda sıcaklığı TSA plakasını etiketleyin veya çubuğu kontrol edin ve ikiye bölün. Zenginleştirilmiş numuneleri inkübatörden çıkarın ve buza yerleştirin. Karıştırmak için örnekleri kısaca girdaplayın, ardından numunenin 10 mikrolitresini TSA plakasına koyun ve bir şerit oluşturmak için plakayı eğin.Bunu iki kez tekrarlayın. Plakaların çizgiyi bölen diğer tarafında, her biri 10 mikrolitre örnekle 10 nokta oluşturun. Tabakları 37 santigrat derecede 18 saat, iki gün ve dört gün boyunca agar plakalarının kurumasını önleyen temiz bir odada kuluçkaya yatırın.Şeritlerdeki kolonileri sayın. Morfolojiye dikkat edin ve morfolojik olarak benzer izolatlar için çubuk başına koloni oluşturma birimlerini hesaplayın. Şeritlerde benzersiz organizmaların yakalanmadığı noktalara bakın.Görselleştirmeyi kolaylaştırmak için bakterileri orta ve sağ plakalarda yoğun bir şekilde kapladık. Tipik olarak, göz bezi plakalarında çok daha az bakteri gözlemledik. Bazen, göz sürüntülerinin geri kazanılabilir bakteriler vermeyebileceğini unutmayın.Burada C57 siyah altı fareden morfolojik olarak farklı izolelere sahip temsili bir göz çubuğu plakası gösterilmiştir. Her ayrı izole için, koloniler şeritte sayıldı ve göreceli bolluk hesaplandı ve çizildi. Mikrobiyolojik karakterizasyon için, bakteriler ana bir TSA plakası üretmek için bireysel fare gözü çubuk plakalarından seçildi.Büyüme ortaya çıktığında, mikropları karakterize etmek veya tanımlamak için ek testler yapıldı. Ana plaka, ilgili izolatları genişletmek için yeterli inokülyum sağlamak için kullanılmıştır. İzoleleri tanımlamak için bir gecede bir TSA plakası çizildi ve inkübe edildi ve MALDI-TOF MS yapıldı.Burada gösterilen izolat örnekleri Streptococcus acidominimus ve Aerococcus viridans olarak tanımlanmıştır. Erkek ve dişi C57 siyah altı fareden önemli ölçüde farklı düzeylerde östensel organizmalar elde edildi. Streptococcus acidominimus, Aerococcus viridans coagulates negatif Staphylococci ve E.coli C57 siyah altı N fareden izole edildi.Bu protokolü denerken, en iyi sonucun çubuğu ince bir şekilde kaplayarak ve göz çubuğu adımı sırasında zaman alarak elde edileceğini unutmayın. MALDI-TOF MS'e erişim sınırlıysa, izolatlar mikrobiyolojik ve biyokimyasal testlerle tanımlanabilir.

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.

Den Beyin sekestre Lökositler İzolasyonu ve Analizi<em&gt; Plasmodium berghei</em&gt; ANKA-enfekte Fare

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

İmmünoloji ve Enfeksiyon

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.

MALDI-TOF kütle spektrometrisi kullanılarak kan kültür besi yerinden, Gram negatif bakterilerin hızlı belirlenmesi

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.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

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.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

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.

Proteinlerin içinde Planta serilerinde Plasmodesmal yerelleştirme tanımlaması

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.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

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.

Kimlik ve yalıtım Oligopotent ve Lineage için kabul edilen miyeloid ataları fare kemik iliği dan

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.

Yalıtım ve kalp Extravascular bağışıklık hücreleri tanımlaması

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.

Bir Galleria mellonella Oral yönetim modeli için Study Commensal-Induced doğuştan gelen bağışıklık yanıtı

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.