Yazarlar cömertçe α-katenin sağlamak için Dr. Le Trinh ve Dr. Tobin teşekkür: sitrin balık hattı ve Bill Jackman bize onun laboratuarında filme yapmak için izin için. Yazarlar fon kaynaklarını Ulusal Sağlık Enstitüleri (Hibeler 5P20RR016463, 8P20GM103423 ve R15AI094406) ve USDA kabul (Proje # ME0-H-1-00517-13). Bu yazının ana Tarım ve Ormancılık Deney İstasyonu yayın sayısı 3371 olarak yayınlanmıştır.

NOT: Tüm Zebra balığı bakım protokolleri ve deneyler Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) protokolü A2012-11-03 altında NIH kurallarına uygun olarak yapıldı. 4 Gün Post Döllenme 1. Zebra balığı Yetiştirme <ol><li> Başka bir video 34 &#39;de gösterildiği gibi, ilk 3 saat sonra döllenme içinde, AB zebrafish veya başka bir transjenik çizgiler toplayın. </li><li> (0.17 mM KCI, 0.33 mM CaCl2, 0,33 mM MgCl2, 2 mM HEPES, pH 6.8, 5 mM NaCl) metilen mavisi ile (0.3 ug / ml nihai E3 ortam 150 ml içeren 15 cm&#39;lik bir petri tabaklarında 120 yumurta inkübe 12/12 açık / koyu fotoperiyodun ile 33 ° C inkübatör konsantrasyon). </li><li> E3 + propiltiyourasil&#39;i (1-fenil-2-tioüre, 10 ug / ml nihai konsantrasyon) ile 6 saat sonra ortam yerine ve ölü yumurta çıkarın. </li><li> 2 gün sonra, taze E3 + propiltiyourasil&#39;i Ortamı alışverişi, 33 ° C&#39;de 4 gün boyunca inkübe edilir. </li></ol> 2. içindejection mikro-iğne hazırlanması <ol><li> Aşağıdaki ayarlarla bir iğne çektirmenin kullanarak bir mikro-iğne içine; borosilikat kılcal (ID 0.69 mm OD 1.2 mm) çekin (550 Heat, 0 çekin, 130 Velocity 110 Saat). NOT: Ayarlar filamanın değişecektir (Şekil 1) benzer şekilli mikro-iğneler elde etmek filament ve yeni bir filaman takıldığında yeniden ayarlanması gerekir. </li><li> 0.4 um filtreden fosfat tamponlu salin 3 ul mikro-iğne ile doldurun (; 137 mM NaCI; 2.7 mM KCI, PBS, 10 mM, Na 2 HPO 4; 1,8 mM KH 2 PO 4, 7.4 pH) 20 ul Microloader pipet kullanılarak ucu. </li><li> Bir basınçlı enjeksiyon sistemine bağlı bir micromanipulator bir mikropipet tutucu mikro-iğne ayarlayın. 30 PSI enjeksiyon basıncını ve 30 msn de darbe süresini ayarlayın. </li><li> Iğne 1/5 su içinde olana kadar damıtılmış su ile bir petri kabı kullanarak, mikro iğne düşük. Mikro-iğne, wi Klipsu seviyesinin hemen altında ince cımbız th. Sudan mikro-iğne getir ve bir bolus görünene kadar pedalına birkaç kez itin. </li><li> Çeşidi bir damla bir daldırma yağı ile katmanlı bir hemositometrede mikro-iğne düşürerek bolus çapını ölçün. İstenilen hacim elde etmek için darbe süresi ayarlayın (Tablo 1). </li><li> Yağda stabil kalır (bolus hacmini artırır eğer basıncı düşürmek ve Kapsül hacmi azalırsa backpressure artırmak) için bolus için backpressure ayarlayın. Her mikro-iğne darbe süresini kaydedin. </li><li> Bir Microloader pipet kullanarak aspire mikro-iğne PBS çıkarın ve enjektör geri yerleştirerek ve sürekli darbe anahtarı çevirerek sol-over PBS sınırdışı. Her mikro-iğne Rezervasyon. </li></ol> 3. Candida Hazırlık <ol><li> Streak Candida albicans kodon-optimize dTomato, GFP, BFP, EOS veya Far ile transformeBir maya peptine dekstroz (YPD) agar (10 g / L maya ekstresi, 20 g / L pepton üzerine steril ahşap takoz kullanılarak donmuş stoktan (-80 ° C hazır% 25 gliserol), kırmızı, 20 g / L dekstroz , 20 g / L ağar; otoklava Petri kapları içinde 25 ml boşaltılmıştır) ve 30 ° C de bir gece boyunca inkübe edilir. </li><li> Steril bir ahşap takoz kullanılarak bir koloni almak ve YPD bir sıvı (10 g / L maya ekstresi, 20 g / L pepton, 20 g / L dekstroz, 5 ml kadar inoküle, Otoklavdan geçirilen ve aseptik 16 x 150 mm cam kültürü, 5 ml bölünebilir borular). </li><li> 60 rpm&#39;de bir silindir tambur içinde 30 ° C&#39;de gece boyunca inkübe edin. </li><li> C. 1 ml toplayın 1.7 ml steril santrifüj tüpü içine albicans kültürü ve transferi. </li><li> Sn bir çift için 5.000 xg&#39;de santrifüj. Süpernatant boşaltın ve iyice steril PBS, girdap 1 ml ekleyin. Iki kez bu işlemi tekrarlayın. </li><li> Steril PBS içinde 1000: 1 ml stok 1 seyreltilmesi ile bir hemositometre kullanılarak kolonileri sayın. </li></ol> 4. Zebra balığı EnjekteSwimbladder <ol><li> Bir 33 ° C kuluçka makinesi içinde, 30 dakika için bir enjeksiyon çanak (% 2 agaroz) ısıtın. </li><li> İstenen zaman, herhangi bir zebrafish aspire olmadan 25 mi pipet kullanılarak enjekte balık içeren 15 cm Petri için E3 + propiltiyourasil&#39;i, 150 ml, 100 çıkarın. </li><li> Geri kalan, 50 ml ortam (nihai konsantrasyon 200 mg / ml) bir 4 mg / ml tampon tricaine metan sülfonat stok (TR) 2 ml ekleyerek zebrabalıkları dpf 4 anestezisi ve 15 dakika boyunca bekleyin. </li><li> Bir mikroskop altında, şişirilmiş bir statik taşıma ile balık seçin. Balık aynı zamanda MPO nötrofil dağıtımı, homojen fenotip için o anda taranabilir: GFP çizgi mikroskop diseksiyon bir Epifloresans kullanarak. </li><li> C seyreltin orijinal konsantrasyon albicans stok 1.5 x 10 7 koloni PBS ml (cfu / ml) oluşturan üniteler için (adım 3.8). </li><li> Plastik bir transfer borusu kullanılarak enjeksiyon çanak üzerine 30 zebrafish aktarıntte ve aşırı ortamı çıkarın. </li><li> Bir balıkçı tel aracı (0.012 inç çaplı olta borosilikat kılcal içine süper yapıştırılmış) kullanarak, dikey enjeksiyon çanak tutarak 10 balık, 3 çizgiler yapmak ve dikey balık yönlendirmek. </li><li> Fazla ortamı çıkarın. </li><li> İyice 1.5 x 10 7 cfu ile tüp vorteks / mL C albicans arasından seçilir. </li><li> C 3 ul bir mikro-iğne doldurun albicans, yeni bir Microloader pipet kullanarak ve uygun ayara darbe süresini ayarlamak (adım 2.9 bakınız). </li><li> Swimbladder (Şekil 3A) arkasına doğru amaçlayan, mikro-iğne ve balık kafası arasındaki 20 ° lik bir açı yapmak için enjeksiyon çanak döndürün. </li><li> , Statik taşıma içine mikro-iğne itin iğne deliği lümen olduğundan emin yapma, ve bir kez pedalına basın. </li><li> Her balık için tekrarlayın ve E3 bir çanak sel bir kurtarma çanak (25 ml E3 + PTU) transferd kurtarma çanak içine boşalma. </li><li> Enjeksiyon çanak başka 30 balık aktarın. </li><li> İyice ile dolu yeni bir mikro-iğne ile enjeksiyon tekrarlayın C vortekslendi albicans arasından seçilir. </li><li> Gerekirse kirlenmesini önlemek ve ayrı bir kurtarma çanak (adım 4.10) aktarmak için farklı bir enjeksiyon çanak kullanarak, PBS kontrol enjekte edilerek bitirin. </li></ol> 5. İstenilen İnokulum için Balık tarama <ol><li> Çözülene kadar mikrodalga ve 37 ° C&#39;ye kadar soğutularak kaynatma, E3 40 ml 0.4% agaroz çözeltisi (LMA) düşük eriyik 160 mg ekleyerek, düşük erime sıcaklığı olan agaroz için 40 mi hazırlayın. Bir kez soğutulduğunda TR (nihai konsantrasyon 200 mg / ml) 400 ul ekle. </li><li> (25 mL, E3 + propiltiyourasil&#39;i, nihai konsantrasyon 200 mg / ml içine TR sadece 1 ml ile adım 4.3) de anlatıldığı gibi, 30 dakika sonrası nekahattan sonra, balık uyuşturan. </li><li> Bir kilo tekne LMA 2 ml (mümkün olduğunca az medya ile) Transfer 30 balık. </li><li> Aspire ayrı bir transfer pipeti ile balık ve sadece 10 x 6 merkez kuyu kullanılarak, 96 çukurlu bir görüntüleme plaka içine LMA&#39;nın 1 damla balık plaka. </li><li> Ekranın tüm balık görüntüleme plakasına yüklenir kadar tekrarlayın. </li><li> Ortam, 5 dakika boyunca oda sıcaklığında katılaşmaya edelim. </li><li> Onlar cam alt ile temas halinde emin, kendi tarafında balık yerleştirin. </li><li> Bir ters Epifloresans / konfokal mikroskop ve uygun filtre üzerinde 20X objektif kullanılarak, C sayısını kaydetmek Her balığın statik taşıma albicans maya hücreleri. </li><li> , Statik taşıma 15-25 maya hücreleri ile balık (veya arzu edilen inokulum) Select ötenazi seçili olmayan balık (E3 TR 800 ug / ml) tarafından E3 + propiltiyourasil&#39;i 50 ml derin Petri kabı seçilen balık dönüş görüntüleme plaka seçilen kuyulara E3 3 damla ekleyerek ve plastik transfer pipet ile balık aspire. </li><li> İstenen süre için 33 ° C&#39;de inkübe edin. </li></ol> 6. Balık Sonrası tarama Görüntüleme <ol><li> İstediğiniz anda, görüntü balık içeren derin Petri kabı E3 + PTU medya 50 ml 25 kaldırın. </li><li> (25 mi, E3 + propiltiyourasil&#39;i kalan, nihai konsantrasyon 200 mg / ml içine TR sadece 1 ml ile adım 4.3) de anlatıldığı gibi balık anestezi uygulayın. </li><li> Uygun bir şekilde 96 oyuklu görüntüleme levhasının ve yerine balık Plate (5,3-5,7 adım). </li><li> Uygun lazerler ve emisyon filtreleri kullanarak konfokal mikroskobu ile görüntü. İlk diferansiyel girişim kontrast (DIC) kapsamında 4X objektif kullanarak balık odaklama ve 2 mikro saniye / piksel hızı ve 1024 x 600 piksel en boy oranı ile tarama modunda konfokal lazerler ile 20X objektif kullanarak ilgi bölgenin görüntülerini elde. 3 kare Kalman filtresi modunu uygulayarak, 1 mikron adım boyutu ile Z-yığınlar edinin. </li><li> 50 ml E3 + PTU veya individua içine derin bir Petri kabı dönl kuyuları (24 bölümlü kültür çanağı, 3 mi E3 + PTU) ile 33 ° C&#39;de inkübe edin. </li></ol> 7. swimbladder Dissecting <ol><li> Yüksek vakum yağ ile bir 10 ml şırınga doldurun. </li><li> % 2&#39;lik bir LMA (agaroz E3 8 ml düşük erime 160 mg adım 5.3) hazırlayın. </li><li> İstediğiniz anda, balık (adım 5.2) uyuşturan. </li><li> Transferi 10 E3, 1 ml ile 1.7 ml santrifüj tüpüne balık ve santrifüj tüpüne 4 mg / ml &#39;Tricaine (nihai konsantrasyon 800 mg / ml) 200 ul ilave edildi ve oda sıcaklığında 15 dakika boyunca inkübe edilerek euthanize. </li><li> Bir mikroslayd (75 x 25 mm, Şekil 2) 1 cm arayla, bir meydanda vakum gres 4 damla koyarak bir görüntüleme cam slayt hazırlayın. </li><li> Meydanın ortasında% 2 LMA 2 damla yerleştirin. </li><li> Bir mikroskop altında ayrı bir cam slayt bir balık yerleştirin ve kalp atışlarımı durduğundan emin olun, aşırı su çıkarmak. </li><lİnce cımbız kullanarak i&gt; sol cımbız ile sabit bir pozisyonda kafa korumak ve doğru cımbız (Şekil 4A) ile ön gut aşağı çekerek swimbladder teşrih. </li><li> Sindirim sisteminden ayıran, sol cımbız ile pnömatik kanal tarafından swimbladder Pick up ve katılaşır önce% 2 LMA merkezine yerleştirin. </li><li> Görüntüleme cam slayt üstüne bir kapak kayma (18 x 18 mm) yerleştirin ve vakum gres yanı sıra, LMA (Şekil 2) dokunana kadar itin. </li><li> Diseksiyon 10 dakika içinde Resim aşama 6.4&#39;te tarif edildiği gibi. Adımı tekrarlayın 7,7-7,11 diğer balık. </li></ol>

The authors have nothing to disclose.

Swimbladder mikroenjeksiyon hastalığı modeli Gelişmeler ve sınırlamaları Burada sunulan model Gratacap et al mukozal kandidiyaz daldırma modelinin bir uzantısıdır (2013).; Bu kontrollü bir enfeksiyon zaman avantaj, yüksek ölçüde tekrarlanabilir enfeksiyon dozu ve bu nedenle geliştirilmiş verimlilik ekler. Biz burada non-invaziv ayrıntılı olarak enfeksiyon dinamikleri zamansal belgeleri yanı sıra statik taşıma yüksek çözünürlük ex vivo g?...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
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		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Mukozal patojenlere karşı savunma Erken bir epitel bariyer ve doğuştan gelen bağışıklık hücreleri, hem oluşur. Her iki immunocompetency ve bunların interkom, enfeksiyonlara karşı korunması için çok önemlidir. Bir patojen ile epitel ve doğuştan gelen bağışıklık hücreleri etkileşimleri en karmaşık davranış zaman ve mekan içinde izlerken vivo, incelenmiştir. Ancak, mevcut modeller mukozal düzeyinde patojenler ile savaş kolay uzay-zamansal görüntüleme için izin vermez. Burada geliştirilen model çocuk zebrabalıkları statik taşıma içine mantar patojeni, Candida albicans, doğrudan enjeksiyon sureti ile bir mukozal infeksiyonu oluşturur. Ortaya çıkan enfeksiyon mukozal hastalık gelişimi boyunca epitel ve doğuştan gelen bağışıklık hücre davranışının yüksek çözünürlüklü görüntüleme sağlar. Bu yöntemin çok yönlülük ph yol açan bağışıklık olaylar ayrıntılı dizisini prob konağın sorgulama için izin veriragocyte işe özel hücre tipleri ve koruma moleküler yolların rollerini incelemek ve. Buna ek olarak, immün bir fonksiyonu olarak patojen davranışı floresan protein ifade C ile eş zamanlı olarak görüntülenebilir albicans arasından seçilir. Konak-patojen etkileşimi artmış uzaysal çözünürlüğü açıklanan hızlı swimbladder diseksiyon tekniği kullanılarak da mümkündür. Burada anlatılan mukozal enfeksiyonu modeli mukozal kandidiazis çalışma için değerli bir araç yapma, basit ve son derece tekrarlanabilir. Bu sistem, aynı zamanda, normal epitel yüzeyleri boyunca enfekte mikobakteriyel bakteriyel veya viral mikropların gibi diğer mukozal patojenlerin geniş çapta çevrilebilir olabilir.

Posterior statik taşıma içinde Mikroenjeksiyon Burada sunulan deneysel yöntem C tutarlı bir doz enjeksiyon açıklar zebrabalıkları dpf 4 statik taşıma albicans maya hücreleri. Daldırma modeli ile Önceki iş önerdiği C swimbladder immün yanıt Candida albicans, memeli mukozal kandidiyazis 32 benzerdir. Burada daha basit, tekrarlanabilir ve hızlı bir modifiye enfeksiyon yöntemi göstermektedir; zebrabalıkları bir kaç yüz enjekt...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Swimbladder Enjeksiyon ile Larva Zebra balığı Mukozal Candidiazis Modelleme

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K Görüntüleme.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Video: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Zebra balığı Larva yılında Dissemine Kandidiyazis non-invaziv Görüntüleme

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

İmmünoloji ve Enfeksiyon

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Tüp bebek Patojen Kaynaklı Nötrofil Trans-epitel Göçünü Değerlendirmek için Coculture Tahlili

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Mukozal astar sıvı noninvaziv örnekleme için In Vivo üst solunum yolu bağışıklık arabulucu düzeyleri miktar

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Enterik patojenler safra tuzu kaynaklı biyofilm oluşumu: kimlik ve miktar teknikleri

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Tüberküloz Mycobacterium marinum enfekte yetişkin zebra balığı içinde modelleme

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Galleria mellonella Waxworm enfeksiyon modeli Dissemine kandidiyazis için

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Bleomisin endotrakeal enjeksiyon tarafından fare akciğer hasarı indüksiyon

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Uyuşturucu Yüklü Lipozomenjekte ederek Larva Zebra balığı Makrofajlarına Yönelik İlaçların Hedeflenmesi

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Bademcikler Mononükleer Hücrelerin İzolasyon Ex Vivo Doğuştan Gelen Bağışıklık Yanıtları Bir İnsan Mukozal Lenfoid Doku Çalışma

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...