Bu çalışma, Kanada Sağlık Araştırma Enstitüsü (CIHR, MOP-86.749) bir araştırma hibe ile desteklendi. L. Liu CIHR Yeni Araştırmacı Ödülü (MSH 95.374) bir alıcı.

1. Agaroz Jel kemoatraktan hazırlanması <ol><li> Pipet 10 50-ml konik tüp 2 × PBS mL tüp ve sıcak su içeren behere koyarak ısınmak. </li><li> Pipet 10 ml distile su ve 50 ml konik başka bir tüp içinde 0.4 g agaroz tozu ekleyin (kap biraz gevşetti) ve karışım ısı (700 watt mikrodalga fırında yaklaşık 1 dakika için) bir mikrodalga fırın sadece kaynama kadar . </li><li> Agaroz çözüm tüp, girdap karma çözüm ısıtılmış 2 × PBS ekleyin ve sıcak su beher sıcak tutmak. </li><li> Mikropipet kemoatraktan 3 ul Hindistan mürekkep içeren 1.5 ml Eppendorf tüp kapağının içine çözümü (örneğin, CXC kemokin 1 mM WKYMVm MIP-2 veya 12 ul 0.5 mikrogram 10 ul) ve (kaçınmak mikropipet kullanarak aspirasyonu ile iyice karıştırın hava kabarcığı). </li><li> 200-ul pipetlemeyin kapağının içine bir ucu ve agaroz çözeltisi (42 ° C) mikropipet 110 ul ucu sonu kesin ve hemen başka bir pipet (balonlu önlemek) kullanarak iyice karıştırın. </li><li> Kemoatraktan içeren jel Mağaza +4 ° C </li></ol> 2. Cremaster&#39;ın kas intravital Mikroskopi için hazırlanması (Şekil 1) <ol><li> 10 mg / kg xylazine ve 200 mg / kg ketamin hidroklorür karışımı bir ip enjeksiyon yoluyla bir yetişkin erkek fare anestezisi. </li><li> Tıraş sağ eksternal juguler ven ve elektrikli tıraş makinesi ile skrotum ön yönü üzerinde alan. Anestezi sonrası, anestezi fare için özel bir dikkat ve bakım vermek önemlidir. Hipotermi fare önlemek için kullanılan bir ısı lambası olabilir. Fare ağrı refleks arındırılmış olmalıdır. </li><li> Yatay bir kesi yapmak, bulmak ve 100 U / mL heparin tuzlu su ile dolu bir PE-10 tüp kullanarak juguler ven catheterize. Juguler ven kateterizasyonu, gerektiğinde ek anestezikler ve ilaçların yönetimi için gereklidir. </li><li> Fare arka ayakları, fare, bir ev yapımı Cremaster&#39;ın kas pansiyon (Şekil 1) yüz kadar yalan göbek bant ile sabitleyin. </li><li> Cremaster&#39;ın kas ve fare vücudu sıcak tutmak için 37 ° C su sirkülatör kurulu bağlayın. </li></ol> Not: 2.6 &#39;den 2.14 Tüm prosedürler, çok nazikçe 5 yapılmalıdır. <ol start="6"><li> Sol Cremaster&#39;ın kas maruz skrotal deride bir kesi olun. Ilgili fasya kas dikkatlice disseke. </li><li> 37 Cremaster&#39;ın kas Superfuse ° C ısıtılmış bir peristaltik pompa kullanılarak (131,9 NaCl, 4.7 mM KCl, 1.2 MgSO 4, 20 NaHCO 3, pH 7.4) tuzlu bikarbonat-tamponlu. </li><li> 4-0 dikiş Cremaster&#39;ın kas kurulu açık görüş cam kaide üzerinde basılı tutun Cremaster&#39;ın kas distal ucuna bağlayın. </li><li> Boyuna Cremaster&#39;ın kas dağlamak. 4-0 sütür ile, kas düz tutun ve kaide üzerinde kenarlarında güvenli. Altta yatan kas testis ve epididim ayrı ve karın boşluğuna taşıyın. </li><li> ~ 37 ° C ısıtılmış perfüzyon tampon ile 0.6 ml / dak ve 22 ile açıkta kalan kas kapağı × 22 mm cam lamel kas Superfuse. </li><li> Mikroskop sahnede Cremaster&#39;ın kas kartı yerleştirin, mikroskop altında kas incelemek uygun bir postkapiller venule (düz ve dalsız ve normal kayma hızı ve çapı 25-40 mikron venule seçin) bulmak ve video kamera ayarlamak venule TV monitörü ya sol ya da sağ ucunda dik bir konumda görüntülenebilmekte izin vermek. </li><li> 30 dakika denge sonra, bir video kaydedici kullanılarak bazal veri olarak 5 dakika süreyle seçilen postkapiller venule video görüntüleri kaydeder. </li><li> Superfusion durdurun ve kas lamel kaldırmak. </li><li> ~ 1 mm 3 boyutlu bir kemoatraktan içeren jel, önceden seçilmiş bir alanda 350 mikron Cremaster&#39;ın kas yüzey üzerine yerleştirin ve paralel gözlenen postkapiller venule, lamel eklemek yerine jel tutun ve çok az kas dokusu superfuse yavaş yavaş piyasaya çıktı ve jel oluşan kemoatraktan bir degrade kurulmasına izin vermek için yavaş hızı (≤ 10 ul / dk). </li><li> Kemoatraktan içeren jel ilave edildikten sonra 90 dakika video görüntü kaydetme. Kayıt sırasında, ayarlamak ve yapıştırma mikroskop odak tutmak, tarama transmigrating ve venule içinde ve kas dokusunda lökosit chemotaxing. </li><li> Deney sonra, analiz için bir bilgisayar video dosyası almak. </li></ol> 3. ImageJ Kullanarak Hücre Takibi <ol><li> Bir bilgisayarda, özü ve AVI formatında (örneğin, AVI dosyası DVD video dönüştürmek için ücretsiz bilgisayar yazılımı bitRipper kullanmak) video dönüştürme. </li><li> Zaman lapsed film üretmek için video düzenleme yazılımı kullanın. Örneğin, bir zaman lapsed film (1 / 512 veya 1 / 1024 &#39;time-lapse olmak için, Windows Movie Maker birorijinal, gerçek zamanlı video t 30-fps oranı). Sıkıştırılmamış zaman lapsed film dönüştürme ve DV-AVI formatında kaydetmek. </li><li> Aynı mikroskop ayarı altında kalibrasyon mikrometre görüntüleri kaydetme, görüntüleri bilgisayara aktarmak, ImageJ görüntüleri açmak. ImageJ, toplam piksel (örneğin, 720 × 480 piksel) ekran sol üst köşesinde görünür. X ve Y eksenleri (örneğin, 200 × 150 mm) hem ekranın boyutunu ölçün. Bu, mikron başına piksel (örneğin, x = 720/200 = 3.6 piksel / mm, ve y = 480 / 150 = 3.2 piksel / mm) sayısını hesaplamak. </li><li> Film, açık ImageJ almak için tekrar &quot;QT Film Açıcı&quot; arayüz &quot;Tamam&quot; Dosya-İthalat-Quicktime Filmleri Plug-in &quot;, analiz edilecek film seçin ve tıklayın. </li><li> &quot;Eklentiler Manuel İzleme&quot; hücreleri izlemek için tıklayın. Başlangıç ​​izleme önce alt alanlarına ilgili bilgileri doldurun. Kısaca, <ol><li> Zaman aralığı (sn) = fold-time-lapse/30 (Örneğin, 1020 × 1020 / 30 = 34 sn / kare aralığını zaman atlamalı olacaktır). </li><li> x / y kalibrasyon = mikrometre görüntü kalibrasyon kullanarak mikron / piksel ölçümü. </li><li> z kalibrasyon = 0 (fare Cremaster&#39;ın kas doku ve hücre taramakta ve göç, son derece ince bir tabaka parlak alan transillüminasyon altında yaklaşık 2 boyutlu olduğu gibi). </li><li> = 1 merkezleme kare boyutu ara. </li><li> Nokta boyutu / Çizgi genişliği / Yazı boyutu: Gerekirse ayarlanabilir. </li></ol></li><li> Bir referans noktası olarak istikrarlı ve net bir nokta seçin. Bu referans noktası, bütün deney boyunca değişmemiş ve sabit kalır herhangi bir açık ve küçük yapısal noktası olabilir. Son kareye ilk referans noktası izlemek ve &quot;End Parça&quot; düğmesini tıklayın ve &quot;Parça Ekle&quot; seçeneğini tıklatın. Veriler otomatik olarak sonuçlar tablo görünür. </li><li> Nötrofil tarama ve göç takip tek tek tıklamayla her çerçeve içinde ortadan kaybolması, doku görünümünü, hücre izlemek ve Microsoft Excel sonuçları tamamlamak ve kaydetmek için &quot;End Track&quot; tıklayın ve &quot;Parça Ekle&quot;. </li></ol> 4. Nötrofil İşe Alım Parametrelerinin Analizi <ol><li> Microsoft Excel dosyasını açın ve verileri analiz (referans noktası değişiklikleri analiz içine almak). </li><li> Intraluminal sürünerek <ol><li> Mesafe Crawling: toplam mesafe hücre optimal göçüne sitesi (mikron) ilk yapışık sitesinden lümen tarar. </li><li> Hız Crawling: uzaktan / saat (mm / dk) tarama. </li></ol></li><li> Transendoteliyal göç <ol><li> Göçüne zaman: hücre, tüm hücre vücut venule dışında hiçbir hücre gövdesi ve lümen (dak veya sn) görülebilir zaman zaman endotel boyunca başka yere başlar zaman. </li><li> Dekolmanı zaman zaman tüm hücre gövdesi, hücre venule (kuyruk retraklar) (dak veya sn) ile teması kesildiğinde, o zaman noktası âlemden sonra (hemen) venule hemen dışında. </li></ol></li><li> Doku içinde Kemotaksis <ol><li> Göç mesafesi: başlangıç ​​noktasından doku içinde göç bitiş noktası (mikron) hücre hamle mesafe toplamı. </li><li> Göç hızı: doku / saat göç mesafesi (mm / dk). </li><li> Kemotaksis mesafesi: miktar mesafe hücre, doku x-ekseni (mikron) geçirir. </li><li> Kemotaksis hızı: kemotaksis uzaktan / saat (mm / dk). </li><li> Kemotaksis göstergesi: doku içinde göç mesafesi kemotaksis mesafe bölme oranı. </li></ol></li></ol> 5. Temsilcisi Sonuçlar: Bightfield intravital mikroskopi lökosit-endotel hücre etkileşimleri çalışma için kullanılır ve nötrofiller için mutlaka olmayabilir rağmen, histoloji çalışmaları, işe hücrelerinin% 95 daha fazla nötrofil kemoatraktan tedavi Cremaster&#39;ın kasları gerçekten nötrofil, doğruladı . Bu raporda, nötrofil seçici chemoattractants kullanarak, in vivo olarak nötrofil işe izleme prosedürleri sunuyoruz. Özellikle, biz nötrofil intraluminal sürünerek izleme, transendoteliyal göç ve zaman atlamalı intravital video mikroskopi ve ImageJ kullanarak anestezi farelerde Cremaster&#39;ın kas dokusu kemotaksis bir protokol açıklar. Cremaster&#39;ın kas kemoatraktan içeren agaroz jel yavaş kemoatraktan bültenleri ve kemoatraktan degrade doku kurulacak sağlar. Nötrofil kemoatraktan farelerde kremasterik postkapiller venüller nötrofil-endotelyal hücre etkileşimleri neden olur. Bütün deney dik bir aydınlık intravital mikroskop altında bir TV monitörü renkli video kamera tarafından projelendirilen ve video kaydedici tarafından kaydedilen video görüntüleri ile birlikte görüntülenmiştir. Biz nötrofil tespitintraluminal sürünerek, transendoteliyal göç ve göç ve yanıt olarak kas dokusunda kemotaksis kemoatraktan MIP-2 nötrofil ve WKYMVm agaroz jel hazırlanan (Şekil 2). MIP-2 (0.5 mcM) ve (0.1 mM) WKYMVm neredeyse karşılaştırılabilir süre, nötrofil göçü ve kas dokusunda kemotaksis venule benzer hızı, nötrofil transendoteliyal göç ve dekolmanı nötrofil intraluminal sürünerek yol açtığını bulundu benzer nötrofil kemotaksisini indeksleri (P&gt; 0.05, Student t testi) ile aynı hız ve. <img src="/files/ftp_upload/3296/3296fig1.jpg" alt="Şekil 1" /> Şekil 1 intravital bir mikroskop sistemi şematik çizimde. Fare Cremaster&#39;ın kas mikroskop sahnede Cremaster&#39;ın kas kurulu açık görüş kaide üzerinde exteriorized ve 37 ° C ısıtılmış bikarbonat-tamponlu salin ile superfused. Dik mikroskop, aydınlık intravital mikroskopi için bir CCD renkli video kamera ile bağlanmıştır. Monokrom derin soğutmalı CCD dijital kamera da, floresan intravital mikroskopi için mikroskop portuna bağlı, görüntülerin hangi doğrudan bir bilgisayar tarafından işlenir. <img src="/files/ftp_upload/3296/3296fig2.jpg" alt="Şekil 2" /> Şekil 2 aydınlık intravital mikroskopi Nötrofil işe parametreleri. Nötrofil göçünü agaroz jel hazırlanmasında MIP-2 veya WKYMVm postkapiller venule bitişik 350 mikron yerleştirilir nötrofil kemoatraktan kademeli olarak salınımı ile sağlandı. Zaman lapsed video veri deney gerçek zamanlı video kayıt işlendikten sonra ImageJ tarafından analiz edildi. Nötrofil intraluminal sürünerek Cremaster&#39;ın kas (D) (A), âlemden zaman ve dekolmanı (B), doku içinde göç hızı ve kemotaksis hızı (C), ve kemotaksis indeksi, MIP-2 veya WKYMVm agaroz jel üzerinde uygulandıktan sonra belirlendi C57BL / 6 farelerde Cremaster&#39;ın kas (n = 3, # paletli hücrelerinin = 22 (A ve B) ve 27 (C ve D) sırasıyla MIP-2 = 26 (A ve B) ve 44 ( WKYMVm sırasıyla C ve D)).

<ol>
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Çıkar çatışması ilan etti.

Intravital mikroskopi inflamasyon sırasında lökosit işe hücresel ve moleküler mekanizmaları ifşa ettiği için önemli bir araçtır. Cremaster&#39;ın kas ve mezenter gibi saydam dokuların mikrodolaşımını lökosit-endotel hücre etkileşimleri tayini için nicel görselleştirme 1,5 tekniğini uygulama için altın standart olmaya devam etmektedir. Geleneksel aydınlık intravital mikroskopi birçok benzersiz teknik özelliklere sahip ve bu dokularda ortaya çıkan işe alma mekanizmaları i...

Tablo: Malzemeler, Özel Reaktifler ve Ekipmanları <table border="1"><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Polietilen boru, PI10 </td><td> Becton Dickinson </td><td> 427401 </td><td> ID 0.28mm x OD 0.61mm </td></tr><tr><td> Çini mürekkebi </td><td> Speedball Sanat </td><td> Süper Siyah </td><td> % 100 karbon siyah pigment-hayır boyalar </td></tr><tr><td> Dağlama </td><td> Aaron Medical </td><td> AA03 </td><td></td></tr><tr><td> Xylazine </td><td> Bayer HealthCare, Bayer Inc. </td><td> DIN 02169592 </td><td></td></tr><tr><td> Ketamin hidroklorür </td><td> Bioniche Hayvan Sağlığı Kanada, Inc. </td><td> DIN 01989529 </td><td></td></tr><tr><td> Murin rekombinant MIP-2 </td><td> Ar-Ge Sistemleri </td><td> 452-M2 </td><td></td></tr><tr><td> WKYMVm </td><td> Phoenix Pharmaceuticals, Inc. </td><td> 072-12 </td><td></td></tr><tr><td> Agaroz </td><td> Invitrogen </td><td> 15510-027 </td><td> Ultrasaf </td></tr><tr><td> Heparin </td><td> Sigma </td><td> H-3393 </td><td></td></tr><tr><td> Dik mikroskop </td><td> Olympus </td><td> BX61WI </td><td></td></tr><tr><td> 3CCD renkli video kamera </td><td> SONY </td><td> DXC-990 </td><td></td></tr><tr><td> HD-DVD video kaydedici </td><td> LG Electronics Inc. </td><td> RH398H-M </td><td></td></tr><tr><td> TV monitörü </td><td> LG Electronics Inc. </td><td> 22LG30 </td><td></td></tr><tr><td> Su sirkülatör </td><td> Thermo Scientific </td><td> HAAKE DC10 </td><td></td></tr><tr><td> Peristaltik pompa </td><td> Gilson; Pharmacia </td><td> Gilson MINIPULS 3; Pharmacia P-3 </td><td></td></tr><tr><td> Cremaster&#39;ın kas kartı </td><td> University of Saskatchewan </td><td></td><td> Ev yapımı </td></tr></table>

tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy

Kan akışından iltihaplı doku lökosit dolaşan işe inflamasyon 1,2 önemli ve karmaşık bir süreç. Iltihaplı doku postkapiller venüller, başlangıçta halata ve rulo venüler duvar luminal yüzeyinde lökosit. Endotel lökosit tutuklama Haddeleme ve kemokin veya venüler yüzeyinde diğer chemoattractants yanıt firma yapışma tabi. Birçok yapışık lökositlerin endotel junctional ekstravazasyonu sitesine yapışma ilk sitesinden taşınmaya, intraluminal 3 sürünen bir süreç olarak nitelendirdi. Taramasını takiben, lökosit kemoatraktan, kaynak (kemotaksis) 4 doğru ekstravasküler doku endotel (geçişe) üzerinden hareket ve geçiş yapma. Intravital mikroskopi, in vivo lökosit-endotel hücre etkileşimleri görselleştirme ve lökosit işe 2,5 hücresel ve moleküler mekanizmaları ifşa için güçlü bir araçtır . Bu raporda, aydınlık intravital mikroskopi kullanılarak nötrofil kemoatraktan degrade yanıt fare Cremaster&#39;ın kas nötrofil göçünü ayrıntılı süreçlerini görselleştirmek ve belirlemek için kapsamlı bir açıklama sağlar. Nötrofil göçünü, nötrofil kemoatraktan MIP-2 (CXCL2, CXC kemokin) veya WKYMVm (Trp-Lys-Tyr-Val-D-Met, sentetik bir analog içeren agaroz jel küçük bir parça (~ 1 mm 3 boyut) ikna etmek için bakteriyel peptid) gözlenen postkapiller venule komşu kas dokusu yerleştirilir. Zaman-lapsed video fotoğraf ve bilgisayar yazılımı ImageJ, endotele nötrofil transendoteliyal göç ve göç ve doku kemotaksis nötrofil intraluminal sürünerek görüntülenebilmekte ve izlenir. Bu protokol intraluminal sürünerek hızı, âlemden zaman, dekolmanı zaman, göç hızı, kemotaksi hız ve doku kemotaksis indeksi gibi pek çok nötrofil göçünü parametrelerin güvenilir ve kantitatif analiz sağlar. Biz bu protokolü kullanarak, bu nötrofil göçünü parametreleri stabil bir şekilde belirlenir ve tek bir hücre lokomosyon uygun in vivo olarak takip edilebilir olduğunu göstermektedir.

Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Biz aydınlık intravital mikroskopi, in vivo olarak nötrofil kemoatraktan kaynak yanıt nötrofil göçünü sırasında dinamik nötrofil-endotel hücre etkileşimleri ölçmek için bir protokol tanımlamaktadır. Nötrofil intraluminal tarama, fare Cremaster&#39;ın kas dokusu transendoteliyal göç ve kemotaksis zaman lapsed video ImageJ ile fotoğraf ve takip ile görüntülendi.

Takip Nötrofil Lumen Crawling, intravital video Mikroskopi Doku transendoteliyal Göç ve Kemotaksis

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

tracking-neutrophil-intraluminal-crawling-transendothelial-migration

Research

JoVE Journal

Immunology and Infection

19.6K Görüntüleme.  University of Saskatchewan. Biz aydınlık intravital mikroskopi, in vivo olarak nötrofil kemoatraktan kaynak yanıt nötrofil göçünü sırasında dinamik nötrofil-endotel hücre etkileşimleri ölçmek için bir protokol tanımlamaktadır. Nötrofil intraluminal tarama, fare Cremaster'ın kas dokusu transendoteliyal göç ve kemotaksis zaman lapsed video ImageJ ile fotoğraf ve takip ile görüntülendi.

Video: Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Nötrofil Ekstrasellüler Tuzaklar: Onları nasıl oluşturun ve görselleştirin için

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

İmmünoloji ve Enfeksiyon

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Kasık lenf düğüm intravital Mikroskopi

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2-Foton Mikroskopi kullanarak Fare Thymus intravital Görüntüleme

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

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 ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

Bir kültür Mesane EPİTELİ karşısında İnsan Nötrofil Göç Kantitatif Değerlendirilmesi

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Germinal Merkezleri derece Çözülmüş Intravital Çizgili-aydınlatma Mikroskopi

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Farelerde mesenteriumda Damarlardaki intravital Mikroskopi Lökosit-endotelyal ve Trombosit-lökosit Etkileşimleri

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Real Time Anestetize Farelerde Intravital Mikroskopi Mezenterik Damarlardaki nötrofiller ve monositler Görüntüleme

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

IL-1β Promoter odaklı DsRed Muhabir Fare kullanma Nötrofil Hazırlama intravital Görüntüleme

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...