The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

NOT: deneyler (NIH yayın, 8. baskı, 2011) &#39;bakım ve kullanım Laboratuvar Hayvanlar olan Kılavuzu&#39;nda&#39; Aşağıdaki hayvan çalışmaları düzenleyen Alman mevzuatına uygun olarak yapıldı ve hayvanların korunması konusunda Direktif 2010/63 / AB Bilimsel amaçlı (Avrupa Birliği Resmi Gazetesi, 2010) için kullanılır. Resmi izin hükümet hayvan bakımı ve kullanımı ofis (LANUV Nordrhein-Westfalen, Recklinghausen, Almanya) verildi. NOT: Kısa vadeli görüntüleme için atlanabilir adımlar hazırlık süresini azaltmak ve cerrahi protokol basitleştirmek için yıldız (*) ile işaretlenmiştir (örn da çekim, 3-D yığınları ya da kısa süreli time-lapse mikroskopi ek). Gerekli Ancak, hayatta kalma süresi büyük ölçüde azalmış olması durumunda Görüntüleme aynı zamanda geniş bir izleme ve sirkülasyon kontrolü olmadan gerçekleştirilebilir. 1. Mikroskop Kurulum ve Ön-cerrahi Hazırlama (5-10 miln) <ol><li> (Mikroskop, güç Lab, lazer, ısıtma yastığı, klima odasının, web cam, şırınga pompası cihazının, solunum) açın sistemi. </li><li> 1.5% Vol Ç 2 Isoflurane Vapor beslemesini ve set İsofluranın seviyesini bağlayın (v / v), küçük bir hayvan solunum cihazına bağlanmak. <ol><li> Kısa süreli görüntüleme için, sadece ilk ip anestezi indüksiyonu sonrası izofluran kullanarak anestezi korumak (adım 2.2). Daha sonra stabil, karaciğer mikro dolaşımı garanti altına almak için hacimce% 2 (hac / hac), izofluran kullanımı, ve 2 saat aşağıdaki görüntüleme zaman tutmak. </li></ol></li><li> 150-200 ul hacminde 120-140 solunum devir / dak solunum ayarlayın. </li><li> Fare agaroz sahne ayarlayın. 40 ° &#39;lik bir açı ile bir çanak (yaklaşık 10 cm x 15 cm, baz) içine dökülerek,% 3 (w / v) agaroz 100 ml hazırlayın. </li><li> Gerekli aletleri toplama cerrahi çalışma alanını ayarlayın. Mikrobiyal enfeksiyonu önlemek için, Sekusept Forte S bir% 1 çözeltisi (kullanarak aletleri% 9.9 w / v dezenfekte) Glutaraldehit w / v% 9.8, deney (Şekil 1A önce 5 dakika için formaldehit). </li><li> Kalan bileşenler elde edilir: Cilt dezenfektan (örneğin,% 10 povidon iyot solüsyonu), karaciğer aşama (yığınları ve köprü), agaroz çözeltisi (% 3 (w / v) PBS içinde)% 5, şırınga pompası (ağ / hac) glikoz çözeltisi anestetik çözeltisi (Ketamin 0.1 mg / ml, iksilazin 0.5 mg / ml, Fentanil 5 ug / ml), pamuklu, n-Butil-2-Cyanoacrylat ve 1x PBS ile (G-5), enjektör pompası olabilir. Ön ısıtma için kuluçka odasına agaroz sahne çanak koyun. </li></ol> 2. Trakeotomi (10-15 dk) <ol><li> 25-28 g ağırlığında ev yetiştiriciliğinde gelen C57BL / 6 fareler kullanın. House, 12 saat aydınlık / 12 saat karanlık çevrimi olan bir ısı- olarak FELASA kılavuzlarına uygun ve nem kontrollü bir ortamda belirli bir patojen içermeyen koşullar altında fareler. Hayvanların standart fareler diyet (koklayın Standart Kemirgen Diyet) ve su ad libitum ücretsiz erişime izin ver. </li><li> Init tarafından fare anestezisiial intraperitonal anestetik II çözeltisi 250 ul (Ketamin 0.1 mg / ml, iksilazin 1 mg / ml, Buprenorfin 10 ug / ml) (İP) enjeksiyon. <ol><li> * Intravital görüntüleme sırasında bakım için, sürekli kesintisiz ip enjeksiyonu ile anestezi çözeltisi I yönetmek (Ketamin 0.1 mg / ml, iksilazin 0.5 mg / ml, Fentanil 5 ug / ml), 0.2 ml / st bir akış oranında bir şırınga pompası cihazı kullanılarak enhalasyon izofluran 0.5% Vol kombinasyonu (hac / hac). </li><li> , Trakeotomi için cildi dezenfekte, 5 dakika (forseps ile örneğin tutam ayak pedi) sonra refleksleri kontrol fare cerrahi hoşgörülü anestezi durumunda ise hazırlık başlar. </li><li> (Şekil 1B) kurumasını önlemek için göz kornea koruyucu krem (örneğin, Dekspantenol 50 mg / g) uygulayın. </li></ol></li><li> Ekstremitelerde için yapışkan bant kullanılarak ventral tarafı açığa hazırlık masaya fareyi sabitleşmek; yavaşça, bu pozisyonda sabitleşmek örneğin., boyun daraltabilir, bir lastik bant kullanarak t bağladımkesici dişler o. <ol><li> Bir pamuklu çubukla dezenfektan uygulayarak, povidon iyot solüsyonu kullanarak cilt ve kürk dezenfekte edin. </li></ol></li><li> Doğrudan çene altına ilk cilt kesme (0.5-1 cm uzunluğunda) gerçekleştirin (Şekil 1C) <ol><li> Dikkatle, tükürük bezlerinin (Şekil 1D) arasındaki bağ dokusunu incelemek. </li><li> Dikkatlice açın kas tüp çevreleyen trakea (Şekil 1E, F) gözyaşı. </li></ol></li><li> Daha sonra havalandırma tüpü sabitleme (Şekil 1G) için trakea altında cerrahi iplik yerleştirin. <ol><li> T-şekilli kesi performans kıkırdak halkaları arasındaki mikro-makas ile Açık trakea (Şekil 1 H) </li></ol></li><li> Insizyon (~ 0.5 cm) ile trakea içine havalandırma tüpü yerleştirin. Ileri tüp itin küçük anatomik forseps ile kaudal ucunu yakala ve yavaşça trakea içine tüp ilerletmek için (Şekil 1I) </li><li> Cerrahi iplik ile tüp sabitleşmek (örn </eiplik ile m&gt;, 5-0) Adım 2.5 trakea içine tüpün etrafında bir düğüm atarak yerleştirilir; Kazara depositioning (Şekil 1J, K) önlemek için cildin borunun ikinci kranial tespiti yapmak. </li><li> Doku yapıştırıcısı (örneğin, n-bütil-2-Cyanoacrylat) (Şekil 1 L) ile sızdırmaz bir kesim. </li><li> Yapışkan bant kullanılarak başında tüp sabitleşmek. </li></ol> 3. laparotomi (15-20 dk) <ol><li> Hipotermi önlemek için ısıtma pedi üzerinde fare yerleştirin. Karın Tıraş, dikkatle deriden saç kaldırmak. Povidon iyot çözeltisi ile traş cilt ve kürk dezenfekte edin. </li><li> Cerrahi makas (Şekil 2A) kullanarak sternum altında kesilmiş küçük bir cilt gerçekleştirin. Kanamayı önlemek için tüm görünür kan damarları dağlama, yanal her iki tarafa da kaburga altında kesim uzatın. </li><li> Dikkatle periton (Şekil 2B) açılış, sternum altında linea alba küçük bir kesim gerçekleştirmek. Cauteriz kullanarak her iki taraf için kesim uzatıntirme (Şekil 2C, D) kanamayı önlemek için. </li><li> Agaroz sahne çanak (Şekil 2E) içine fare yerleştirin. Fare (genellikle 12-14 kapak slipleri) (Şekil 2F) her iki tarafına da, uygun yükseklikte yığınları yerleştirin. </li><li> Solunum ile mikroskop eşitlemek için sırtın üst solunum altında tetik sensörü yerleştirin. Tetik ünitesi (Şekil 2G) etkinleştirin. </li><li> Kaburga (Şekil 2I) geri çekilmesi için sternum ile cerrahi iplik (5-0) yerleştirin. </li><li> Dikkatle kavisli cerrahi makas kullanarak karaciğer ve diyafram (falsiform ligament) bağlayan bağ yanı sıra karaciğer ve gastrointestinal sistem kesti. Aorta (Şekil 2J) aşağı falsiform ligament kesin. </li></ol> 4. Numune Kur (10-15 dk) <ol><li> Büyük karaciğer lobunda kolay erişim için 45 ° &#39;lik bir açı ile sağ tarafta fare yerleştirin. </li><li> Karın kapsayan, kaburga altında evreleme köprü eklekavite. Keskin kenarları (Şekil 2K) kapsayacak şekilde yapışkan bant kaplama standart bir kapak kayma kullanarak bir köprü imalatı. </li><li> Sahnede büyük karaciğer lobu yerleştirin. Dikkatle çift bilyeli kalem probu veya karaciğer altında yastıklı spatula kayma ve ıslak pamuklu bir bez veya ıslak mendil kullanarak organın üst tutun. Slayt üzerine lob kaldırın ve nazik sürükle uygulayın. Lob bükülmüş ya da katlanabilir. Karaciğer (Şekil 2L) sadece nazik manipülasyon için ekstra bakım sağlayın. </li><li> Örneğin, küçük bir kapak fişleri (20 mm x 20 mm), yanındaki karaciğer lobunun yaklaşık aynı yükseklikte yığınları kapak kayma desteklemek için yanal destek hisselerini, yerleştirin. </li><li> Karaciğer lobda büyük kapak kayma (24 mm x 50 mm) yerleştirin. Bu kapak kayma olun mümkün (Şekil 2M) olarak yatay olarak aydınlatmaktadır. NOT: kapak kayma sıkarak olmadan doku ile temas halinde olmalıdır. Engelli kan akımı (beyaz doku) gözle görülür kontrol edin. Microcirc Eğerulation bozulur, daha kazık destek ekleyin. </li><li> * Uzun süreli anestezi ve G-5 uygulama için yer iki bağımsız periton kateterler (Şekil 2N). Bir sonraki arka bacaklarda düşük karın yanal kateter takın. NOT: periton kateterler self-made esnek silikon tüp (Şekil 3) ile 27 G iğne bağlayarak olabilir. </li><li> * Cilde 5-0 cerrahi iplik bir döngü ile sabitleşmek iğne yanlışlıkla değiştirmesini önlemek için. </li><li> * Ben 1 katatere anestezi çözümü ile şırınga pompası takın, 0.2 ml / saat ayarlanmış akış hızı; 0.1 ml / saat kateter 2, G-5, şırınga pompası, sabit akış hızı ekleyin. </li></ol> 5. Fare İzleme <ol><li> * Yeri EKG ön içine elektrotlar ve arka ekstremite (Şekil 4A). </li><li> * 2 seviye sensörü CO son tüp takın. </li><li> (Şekil 4C ısı yastığı bağlı harici sıcaklık sensörü ekle). </li></ol> 6. gömülmesi ve Doku Sabitleme (5-10 dk) <ol><li> 3,% 1 X PBS içinde (ağırlık / hacim) agaroz 100 ml hazırlayın. Sıcaklık 5 ml&#39;lik bir şırınga ve bir 18 G iğne (Şekil 5A) ile 41 ° C &#39;de iken karaciğer gömer. </li><li> Lamel ve fare (Şekil 5B, C) ​​etrafında kalan agaroz dökün. Agaroz tamamen Jelatinize kadar bekleyin. </li><li> Hazırlanan karaciğer lobu (Şekil 5D, E) taramak için yeterli bir viewfield büyük hazırlanması, Heidemann spatula ile fazla agaroz çıkarın. </li></ol> 7. Görüntüleme <ol><li> Mikroskop iklim odasına fare aktarın. </li><li> 1x PBS 50-100 ml ekleyin (37 ° C ısıtılmış). </li><li> Buharlaşma (Şekil 5F) önlemek için numune çanak örtün. </li><li> * 0.5 hacmin% enhalasyon izofluran azaltın (hac / hac). </li><li> * Izleme yazılımı EKG, kalp hızı ve ekspiratuar CO 2 kaydetmeye başlayın. </li><li> Görüntüleme başlatın: la açmakser panjurlar, ilgi alanlarını belirlemek görünüm Z-yığınları için üst ve alt sınırını tanımlamak, ve zaman atlamalı kayıt başlar. Z-sürüklenme düzeltmek için gerekirse (Z-yığınlarının yeniden ayarlayın, örneğin. Sebebiyle kan basıncı ya da sıcaklık dalgalanması, Şekil (5G) değişikliklere. NOT: Hayvanların dolaşımı veya anestezi görüntüleme dönemi veya eğer tamamlanması kararsız hale geldikten sonra, (anestezi uyanış olmadan), boyunları kırılarak fareler kurban. </li></ol>

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The authors disclose no con&#64258;ict of interests.

Bu çalışmanın amacı, karaciğer intravital TPLSM görüntüleme için yüksek ölçüde standardize edilmiş stabil ve tekrarlanabilir bir yöntem geliştirmektir. Genel Intravital görüntüleme hedeflemesi ve geliştirme, homeostasis ve hastalığın farklı lökosit popülasyonlarının etkileşimi aşağıdaki gerçek hayat koşullarında hücresel davranış içine değerli anlayışlar verdi. Ancak, doğrudan diğer katı organlara kıyasla karaciğer yanı sıra yüksek kırılganlık aktarılır hangi neden...

hücre ve bütün organlarda hücresel fonksiyonlar ya da hatta bütün bir organizma görselleştirme gövde 2&#39;nin hemen hemen tüm parçaları dahil olmak üzere, 50 yıldan fazla bir büyük bir ilgi olmuştur. Bu nedenle, bazı erken çalışmalar zaten karaciğer 3,4 intravital görüntüleme istihdam. Ancak, çeşitli sınırlamalar karaciğer dokusunun uzun vadeli istikrarlı yüksek çözünürlüklü görüntüleme ile ilgili güncel var. Nedeniyle diyafram ve gastrointestinal sistem 5 ile yakın temas içinde karaciğer anatomik pozisyonuna, mikroskobik intravital görüntüleme için en yaygın sorun, bağırsak 6 daha az bir ölçüde, peristaltik hareket solunum nedeniyle ve. Diğer katı organlara kıyasla, karaciğer cerrahisi özellikle zordur. Nedeniyle yoğun mikrovasküler yapı, cerrahi manipülasyon, masif hemorajik lezyonlar ikamet bozulmuş mikrosirkülasyon 7 ve ayrıca aktivasyonunu yol açabilir iBu tür Kupffer hücrelerinde 8 olarak mmune hücreleri. Bu nedenle, doku mekanik tespit 6,9 intravital mikroskopi görüntüleme ile müdahale muhtemeldir yerde yayınlanan. Sağlıklı bir karaciğer toplam kan hacminin% 10-15 karaciğer damar içinde bulunduğu, ve organ dolaşımdaki değişiklikler (örneğin, kan basıncı dalgalanmaları oldukça duyarlı bir organ oluşturma, genel kalp debisi 10 civarında% 25 alır ). Bu nedenle, aşırı doku işleme veya merkezi dolaşımı yoluyla örneğin, kayma gerilmesi, yerinden, yaralanma hepatik kan akımı aksamalar yanı sıra karaciğer bağışıklık yanıtları etkileyen, lökosit göç davranışı yapay değişiklikler, bozulmuş karaciğer oksijenlenme ve bu nedenle daha fazla karaciğer hasarına yol açacaktır organ koruyucu ve hayvanın genel yaşam süresidir. Erken mikroskobik çalışmalar Intravital epifloresans mi dayanmaktadırcroscopy, ancak böyle bir fotoğraf ağartma ve düşük penetrasyon derinliği gibi çeşitli teknik kısıtlamalar uzun vadeli karaciğer görüntüleme 4,11,12 için bu tekniğin kullanımını sınırlamak. Bu yeni yöntem, gerçek yaşam durumları 13-15 altında hemen hemen tüm organlarda görüntüleme çalışmaları gerçekleştirmek için teknik yetenekli olduğu gibi 1990&#39;larda multiphoton mikroskopi gelişmesiyle birlikte, fotoğraf beyazlatma veya penetrasyon derinliği sınırlamalar çoğunlukla, çözüldü. Ancak, karaciğer görüntülemesi açısından ana kalan zorluklar vardı: birkaç saat 16 daha uzun süre hepatik Sinüsoidlerde değiştirilmemiş kan akışını ve özellikle kararlı görüntüleme güvence nefes hareketleri, karaciğer dokusunun otofloresansı. Çeşitli çalışmalar karaciğer 17, örneğin, NKT hücreleri 18-20, T hücreleri 21,22, karaciğer makrofajlar 23,24 veya nötrofiller 25, uzun vadeli multiphoton m fonksiyonu ve çeşitli lökositlerin göçünü ele rağmenicroscopy görüntüleme henüz başarılı nedeniyle daha fazla hasara 26 mevcut hasar ve dolayısıyla daha yüksek duyarlılığa bir görev daha zorlu akut veya kronik karaciğer hastalığı olan hayvanlarda, kurulmuş olmasaydı. Ancak, gerçek zamanlı olarak karaciğerde lökositlerin göç davranışlarını ve hücresel fonksiyonu izleme karaciğer homeostazı ve hastalık 27 kendi özel rolü yeni bakış açıları sağlar. Kemokin reseptörü CXCR6, doğal öldürücü (NK) hücreler, NKT hücreleri ve bazı T hücresi popülasyonları 18,28 dahil olmak üzere birçok lenfosit alt, ifade edilir. Farelerde Önceki çalışmalar CXCR6 ve aynı kökenli bağ CXCL16 homeostazı karaciğer sinüsoidlerinden üzerinde NKT hücrelerinin devriye kontrol olabileceğini göstermiştir. Sonuç olarak, (CXCR6 lokusunda yeşil floresan protein [GFP] bir knock-in taşıyan) CXCR6.gfp farelerin kullanılması, beyin 29 gibi çeşitli organ lenfosit göçünü araştırmak için tarif edilmiştirve ayrıca karaciğer 18,20, enflamasyon üzerine CXCR6.gfp hücrelerin infiltrasyonunu artış göstererek. Bu çalışmada verilen yöntemle stabilize koşullar altında uzun bir süre boyunca, bu işlemleri takip edilmesi mümkün olmuştur. Intravital multiphoton bazlı prosedür hayvan ve organın minimal pertürbasyon ile son derece tekrarlanabilir oldu görüntüleme izin; solunum ve dolaşım yakın kontrolü ardından geniş izleme uzun süreli hayvan yaşam için optimize edilmiş; ve son derece esnek ve kolay gibi böbrek veya dalak gibi diğer parankimal organlarda da benimsemeye.

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
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			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
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			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
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			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
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			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
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			<td style="width:263px;"></td>
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			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
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			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
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			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
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			<td style="width:219px;">50-1150</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
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			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
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			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
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			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
		</tr>
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			<td height="21" style="height:21px;width:411px;">Leukoplast</td>
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			<td height="21" style="height:21px;width:411px;">Microscope Slides</td>
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			<td style="width:219px;">1879</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
			<td style="width:263px;">Antiseptica GmbH</td>
			<td style="width:219px;">72PAH200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Tissue paper</td>
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			<td height="21" style="height:21px;width:411px;">Surgical Instruments</td>
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			<td style="width:219px;">S-01134</td>
			<td></td>
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			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
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			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
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			<td style="width:219px;">11151-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Graefe forceps straight x2</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11050-10</td>
			<td></td>
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			<td height="21" style="height:21px;">Heidemann spatula HD2</td>
			<td style="width:263px;">Stoma</td>
			<td style="width:219px;">2030.00</td>
			<td></td>
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			<td height="21" style="height:21px;width:411px;">Needle holder Mathieu</td>
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			<td style="width:219px;">12010-14</td>
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			<td height="21" style="height:21px;width:411px;">Scissor</td>
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			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14029-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">11000-12</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
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			<td height="21" style="height:21px;">Equipment</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature sensor</td>
			<td style="width:263px;">comercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled System for Microscopes -Cube&#38;Box</td>
			<td style="width:263px;">Life Imaging Services</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html> Yaralanmaya yanıt olarak karaciğer iltihabı, monositler, nötrofiller, T hücresi alt kümelerinin, B hücreleri, doğal katil (NK) ve NKT hücreleri de dahil olmak üzere, lökositlerin farklı alt süzmeyi söz konusu eden bir yüksek dinamik bir süreçtir. Bağışıklık hücre göçü izlemek için karaciğer intravital mikroskopi, numune hazırlama ve fiksasyon, optik çözünürlük ve uzun süreli hayvan yaşam konusunda yüksek gereksinimleri özellikle zordur. Ancak, inflamatuar süreçler yanı sıra hücresel etkileşim çalışmaları dinamikleri daha iyi inflamatuar karaciğer hastalığı başlatılması, ilerlemesini ve gerileme anlamak için kritik bilgiler verebilir. Bu nedenle, son derece hassas ve güvenilir bir yöntem yoğun bakım ile kombinasyon halinde göç ve intravital iki-fotonlu lazer tarama mikroskobu (TPLSM) uzun bir süre (yaklaşık 6 saat) fare karaciğer, farklı bağışıklık hücreleri, hücre-hücre etkileşimini incelemek için kurulmuştur izlenmesi. esi &quot;&gt; Resim yöntem yumuşak bir hazırlama ve organın en az pertürbasyon ile karaciğerin stabil bir sabitleme içeren, uzun süreli intravital görüntüleme izleme sağlayan, en fazla 6 saat arasında bir zaman dilimi boyunca hemen hemen hiçbir ışıkla ağartma ya da fototoksik etkileri ile çok renkli multiphoton mikroskopi kullanılarak ve fare hayati parametreleri ve dolaşım, sıcaklık ve gaz değişimi istikrar geniş izleme nedeniyle istikrarlı görüntüleme koşulları, özel lökosit alt gruplarının. CXCR6.gfp karaciğer iltihabı üzerine lenfosit göçünü araştırmak knock-in farelerde bazal şartlar altında ve karbon tetraklorid, periton içine enjekte (ler) i (CCl4) neden olduğu akut ve kronik karaciğer hasarı sonrası intravital karaciğer görüntüleme tabi tutuldu. Doğal Katil (NK) - - ve aynı zamanda, CD4 T hücreleri gibi T lenfositlerinin alt grupları fakat mukozal Associ CXCR6 esas Doğal Katil T (NKT) lenfositlerin eksprese edilen bir kemokin alıcısı, birated değişmez (MAIT) T hücreleri 1. Bir karaciğer hasarı üzerine kendi değişmiş davranış içine ayrıntılı fikir ve hastalığın ilerlemesinde dolayısıyla potansiyel katılımı izin CXCR6.gfp + bağışıklık hücrelerinin göç desen ve konumlandırma ardından.

Bizim Intravital TPLSM yaklaşımı doğrulamak için, biz intravital TPLSM görüntüleme CXCR6 GFP / + fareler tabi. Fareler, ya temel kontrol olarak tedavi edilmeden bırakıldı ve akut karaciğer hasarı 20 uyarılması için karbon tetraklorür, tek bir intraperitoneal enjeksiyon (CCl4) tabi tutuldu. Nedeniyle yeşil floresan Video dizileri 2-5 saatlik bir süre boyunca alındı ​​ve hücreler, zaman içinde takip edildi. Genel hücresel hareketlili...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp Muhabir Fare kullanma Sağlıklı ve hastalıklı Karaciğer Bağışıklık Hücreleri Uzun Vadeli intravital multiphoton Mikroskopi Görüntüleme

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K Görüntüleme.  RWTH University-Hospital Aachen. Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Video: Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

İmmünoloji ve Enfeksiyon

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Uzun Vadeli Kronik<em&gt; Pseudomonas aeruginosa</emFarelerde&gt; Havayolu Enfeksiyon

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Aşağıdaki Karaciğer intravital Mikroskopi Görüntüleme<em&gt; Leishmania</em&gt; Enfeksiyon: Karaciğer Hemodinami Üzerine Bir Değerlendirme

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analizi<em&gt; Yersinia enterocolitica</emAna Hücreleri içine&gt; Efektör Translokasyon Beta-laktamaz Effector Fusions kullanma

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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.

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

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 ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Mycobacterium tüberküloz ile muhabir enzim Floresans farelerde görüntüleme

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...