Bu çalışma Ulusal Sağlık Enstitüleri ile hibe (JC P30 HL101302 ve RO1 HL109439) ve Amerikan Kalp Derneği (JC SDG 5270005) tarafından kısmen desteklenmiştir. A. Barazia bir T32HL007829 NIH eğitim hibesi ile desteklenmiştir.

1. Intravital Mikroskop hazırlanması (Şekil 1A) <ol><li> (125 mM NaCI, 4.5 mM KCI, 2.5 mM CaCl2, 1 mM MgCl2, 17 mM NaHCO 3, pH 7.4) tampon superfusion hazırlayın. </li><li> 37 tampon sıcaklık ve bir termo-kontrollü battaniye korumak için bir dolaşım su banyosu açın ° C. Azot gazı (% 5 CO 2, nitrojen ile dengeli) ile tampon havalandırınız. </li><li> Mikroskop sistemi açın (Sutter Lambda DG-4 yüksek hızlı dalga değiştirici, iş istasyonu bilgisayar, Olympus BX61W mikroskop, MPC-200 çoklu manipülatör, ROE-200 sahne denetleyici, yüksek hızlı kamera ve yoğunlaştırıcı). </li><li> Vasküler inflamasyon modeli için,% 100 dikroik ayna kullanın. Lazer yaralanma ile trombüs oluşumunu teşvik etmek için, 50/50% ayna ile% 100 ayna yerini ve Micropoint lazer ablasyon sistemi açın. </li></ol> 2. Intravital Mikroskopi Cremaster&#39;ın kas hazırlanması (Şekil 1B) <ol><li> Bir uyuşturketamin (125 mg / kg vücut ağırlığı (BW)) ve atlara ip enjeksiyonu (12.5 mg / kg BW) göre erkek fare (6-8 hafta, eski C57BL / 6). Illinois Kurumsal Hayvan Bakım ve Kullanım Kurulu University tüm hayvan bakımı ve deney prosedürleri onayladı. Bir vasküler inflamasyon modeli için (cerrahi öncesi 2-2.5 saat) önceden görüntüleme intrascrotally bir fare 3 saat içine fare TNF-α (250 ul serum fizyolojik içinde 0.5 mg) enjekte. </li><li> Bir intravital mikroskop tepside 37 ° C&#39;de bir termo-kontrollü battaniye üzerinde fare yerleştirin. </li><li> Forsepsi kullanarak boyun yüzeyinde deri çekin ve yukarı makas ile 1 cm yatay boyun deri orta hat kesilirken. </li><li> Yavaşça künt makas kullanarak servikal kas açın ve trakea çevreleyen kas kaldırın. </li><li> Solunum güçlüğü ortadan kaldırmak için trakea içine bir PE90 tüp kanüle. </li><li> Nazikçe servikal kas sol tarafında kaldırmak ve çevre dokulardan juguler ven izole. </li><li> C annuantikorlar ve ek anestezik infüzyonu için juguler ven içine geç PE10 tüp. </li><li> Yavaşça çekin ve yatay olarak skrotal deride kesilirken. Deney boyunca doku bütünlüğünü korumak için, önceden ısıtılmış tampon cerrahi alan üzerinde superfused edildi. </li><li> Tek forcep ile alt karın üzerine basın ve dikkatle diğer forcep ile testis çekin. </li><li> Testis etrafında çevreleyen bağ dokuları çıkartın. </li><li> Dikey cremaster kas kesilirken ve çevre iğneleyerek bir intravital mikroskop tepsi üzerinde bir cam lamel üzerinde kas düzleştirin. </li><li> Veri toplamadan önce stabilize kası için 10-20 dk bekleyin. 30-45 um arasında bir çapa sahip Cremaster&#39;ın kas arteriyoller (trombüs oluşumunu) ve venüllerin (damar iltihabı) çalışma için seçilmiştir. </li><li> Anestezi koşulları sağlamak için, aynı anestezi juguler cannulus yaklaşık olarak her 30 dk 30-50 ml demlenmeye. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 3. TNF-α-ile indüklenen vasküler enflamasyon için intravital Mikroskopisi <ol><li> Intravital mikroskop, açık SlideBook 5.0 yazılımı üzerinde fare yerleştirilmesinden sonra ve uygun optik ve objektif (60X) ayarlamak için menü çubuğunda &quot;Odak Pencere&quot; üzerine tıklayın. </li><li> Dylight infüze 488-konjüge sıçan anti-fare CD42c (100 ul tuzlu su içinde 0.1 mg / g BW) ve Alexa Fluor 647-konjüge sıçan anti-fare Gr-1 antikor (100 ul tuzlu su içinde 0.05 mg / g BW) bir boyun cannulus sayesinde . </li><li> Menü çubuğuna gidin ve araç çubuğundaki &quot;Görüntü Yakala&quot; üzerine tıklayın. </li><li> Yeni bir menü açılır; &quot;Resim&quot; olarak, yüksekliği &quot;460&quot; 1x1 gibi &quot;Bin Faktörü&quot; seçeneğini seçin ve &quot;640&quot; width ve. </li><li> &quot;Filtre Seti&quot; olarak, her biri için poz süresini teşhir ve ayarlamak istediğiniz kanallar için onay kutusunu işaretleyin. Örneğin, açık kanal: 10 msn, FITC kanal: 50 msn ve Cy5 kanal: 50 msn. </li><li> Zaman noktalarında ve aralık sayısını ayarlamak için &quot;Time-Lapse Yakalama&quot; seçeneğini seçin. For inflamasyon modelinde, zaman noktalarının sayısını 200 msn lik bir aralıkla (: 5 dk Toplam geçen zaman) ile 1000-1500 ayarlanır. </li><li> O kanal için belirlenen pozlama seferde tek bir düzlem görüntüyü görmek için &quot;Test&quot; i tıklayın. Histogram yeterli yoğunluk dağılımı (çok kanallı yakalama için, her bir kanal için bu işlemi yineleyin) gösterene kadar maruz kalma süresi ve / veya mikroskop ışık ayarlayın. </li><li> Bir kez tüm parametreleri &quot;Görüntü Yakala&quot; in (kanalları, pozlama süreleri, vb) ayarlanır, yakalama başlamak için &quot;Başlat&quot; seçeneğini seçin. </li><li> Vasküler enflamasyon modelinde bir iltihaplı cremaster venül üst yarısı içinde 5 dakika boyunca haddeleme ve yapışkan trombositler ve nötrofil izler. Sekiz ila on farklı venüllerde bir fare kaydedilir. </li><li> Görüntüleri 157 mikron x 118 mikron bir pencere boyutu veren 60X toplam büyütme (1.0 NA su immersiyon objektif) kullanılarak alınmıştır. </li><li> Deney tamamlandıktan sonra, fareler servikal dislocatio ile ötenazin. </li></ol> 4. Venüler İnflamasyon Modeli için Veri Analizi <ol><li> SlideBook 5.0 yazılımı açın ve bir dosyayı açmak için menü çubuğunda &quot;Klasör&quot; tıklatın. </li><li> Açılan kanal menüsünü tıklayın ve &quot;Aç, FITC, Cy5, vb&quot; seçeneğini </li><li> &quot;(Kırmızı ve yeşil çubuklar) Renormalize&quot; ve kanalı seçin tıklayın. Kabaca floresans sinyali optimize sola veya sağa kırmızı ve yeşil çubuklar sürükleyin. </li><li> &quot;Uygula&quot; ve &quot;Tamam&quot; görüntüyü güncellemek için tıklayın. </li><li> Varsayılan olarak geçerli ekran seçmek için &quot;Küçük Düğme&quot; tıklayın. </li><li> Yakalanan timelapse görüntü oynayın ve 5 dk süresince haddeleme ve yapışık nötrofil say. </li><li> Trombosit trombüs oluşumunu analiz etmek, &quot;Maske&quot; gidin ve &quot;Oluştur&quot; u seçin. </li><li> Bir maskeyi ve &quot;mevcut görüntü olarak&quot; işareti adlandırın. </li><li> Ana görünümünde &quot;Marquee Tool&quot; in &quot;Büyük kalem simgesi&quot; tıklayın. </li><li> Bir arka plan maske ayarlamak için ana görünümünde geminin dışındaki bir bölge renklendirin. </li><li> T gito &quot;Maske&quot;, &quot;Bu Düzlem Kopyala&quot;, &quot;mevcut timepoint Kopyala maske&quot; tıklayın ve &quot;tüm zaman noktalarında&quot; seçeneğini tıklayın ve &quot;Tamam&quot; a tıklayın. </li><li> Arka plan sinyalini hesaplamak için, &quot;İstatistik&quot; gidin ve &quot;Maske İstatistikleri&quot; butonuna tıklayınız. </li><li> Görüntü Kapsam &quot;Cari 2D Time Lapse veya 4D Resim (ler)&quot; tıklayın ve Maske Kapsamında &quot;Tüm Maske&quot; seçeneğini seçin. Yakalama tarihi altında, &quot;Alan (piksel)&quot; Morfometri altında Yoğunluğu altında &quot;Maksimum Yoğunluk&quot; seçeneğini seçin ve &quot;Export&quot; (Bu istatistik yöntemini tıklatın bize sağlar Özellikler olarak, &quot;Geçen Zaman (: mm ms hh)&quot; seçin oto-floresans yoğunluğu (arka plan sinyali)) hesaplamak. </li><li> MS Office Excel metin dosyasını açın ve kayıt dönemi boyunca arka sinyalin ortalama değerini hesaplamak. Bu, arka plan floresans şiddetidir. </li><li> Trombüs yoğunluğunu belirlemek için, ana görünümde &quot;Marquee Tool&quot; tekrar &quot;Büyük kalem simgesi&quot; tıklayın. </li><li> Trombosit sinyalini ayarlamak için ana görünümünde kabın içine Renk. </li><li> &quot;Maske&quot; Git, &quot;Bu Düzlem kopyala&quot;, &quot;Tüm zaman noktalarında&quot; select &quot;Geçerli timepoint Kopyala maskesi&quot; seçeneğine tıklayın ve &quot;Tamam&quot; tıklatın. </li><li> &quot;İstatistik&quot; gidin ve &quot;Maske İstatistikleri&quot; butonuna tıklayınız. </li><li> Görüntü Kapsam &quot;Cari 2D Time Lapse veya 4D Resim (ler)&quot; tıklayın ve Maske Kapsamında &quot;Tüm Maske&quot; seçeneğini seçin. Özellikler olarak, seçme &quot;Geçen Süre (ss: dd: ms)&quot; Capture Tarih altında, bize izin &quot;Alan (piksel)&quot; Morfometri altında Yoğunluğu altında &quot;Sum Yoğunluğu&quot; seçin ve &quot;Export&quot; (Bu istatistik yöntemini tıklatın seçin ) kabın içine floresans yoğunluğunu hesaplamak. </li><li> MS Office Excel metin dosyası açın. Trombositlerin Floresans sinyalinin geminin içindeki toplamı yoğunluğu arka plan yoğunluğu x Bölgesi ortalama değeri (piksel) çıkarılarak hesaplanır. Bu, trombosit trombüs floresans şiddetidir. </li><li> 3-5 farklı farelerde 30 farklı venlerde bu işlemi tekrarlayın. Sonra, trombositlerin floresans şiddetinin ortanca değerini hesaplamak. </li><li> Karşınötrofil yuvarlanma ve adezyon analiz, nötrofil haddeleme akını her venül 5 dk içinde belirlenir ve dakikada hücre sayıları olarak sunuldu. &gt; 30 saniye için durağan ve yavaşça sürünerek (&lt;10 mikron üzerinde 30 sn) ama üzerinde atmadım yapışık nötrofil sayısı, sayılan ve 5 dk hücre başına sayı olarak sunuldu. </li></ol> 5. Lazer kaynaklı Arteriolar Tromboz için intravital Mikroskopisi Ablasyon lazer 5-1 Kalibrasyon <ol><li> Mikroskop sahne üzerine yansıtılmış bir slayt yerleştirin, slaytın üst distile su küçük bir damla uygulamak ve yoğunluğunu ayarlayın ve mercek kullanarak odaklanın. </li><li> &quot;Odak Pencere&quot; açın ve SlideBook 5.0 yazılımı &quot;FRAP&quot; sekmesini seçin. </li><li> 40-50 lazer güç ayarlama, hem de &quot;çift tıklayın tekrar&quot; ve &quot;çift tıklayınız boyutu&quot; 8 ayarlanır. Crosshair&#39;i bir dizi getirmek için &quot;Yerler&quot; kutusunu işaretleyin. </li><li> Görev o gelen &quot;Ok&quot; simgesini seçinn Ekranın üst. </li><li> &quot;FRAP Hizalama&quot; sekmesi altında, &quot;bir sonraki Fire&quot; ı tıklatın. Küçük bir nokta monitörde sol alt köşesine yakın görünmelidir. Nokta göründüğünde, çift bunun ortasına tıklayın. Bir kez tıklandığında, başka bir nokta üstünde belirir ve ilk nokta sağında olmalıdır; bunun üzerine çift tıklayın. 16 puan (16 inci nokta sağ alt köşesinde görünecektir.) Kalibrasyon parametrelerini güncellemek tamamlandığında &quot;Kaydet&quot; düğmesini tıklatın için bu işlemi tekrarlayın. </li><li> Kalibrasyon doğruluğunu sınamak için, ekranın ortasında görünmelidir &quot;Merkezi&quot; düğmesi-küçük bir nokta tıklayın. Değilse istenen doğruluk elde edilene kadar, kalibrasyon tekrarlayın. </li></ol> 5-2 Lazer kaynaklı arterioler trombüs oluşumu <ol><li> Mikroskop sahnede bir anestezi fare (2-2 2-12 yordama bakın) yerleştirin. </li><li> &quot;Yakalama&quot; penceresi altında bir protokol seçin veya yeni bir tane oluşturun. Ayarları aşağıdaki gibidir: CY5 (70 msn maruz kalma), Açık (10 msec maruz kalma), ve FITC (50 msn maruz kalma). Görüntü 20 dk (500 msn bir zaman aralığı ile yaklaşık 2.400 kez puan) üzerinde yakalanır. </li><li> Dylight yukarıda tarif edildiği gibi 488-konjüge fare anti-CD42c ile Alexa Fluor 647-konjüge fare anti-Gr-1 antikor süzülür. </li><li> Olarak 3-7 3-3 açıklanan floresan yoğunluğu ve parlak saha görüntü dahil mikroskobu parametrelerini optimize. </li><li> Uygun aydınlık şiddeti veya antikor sinyal, arteriyol yerleştirme ve odak sağlamak için &quot;Test&quot; düğmesine tıklayın. </li><li> Görüntü yakalama işlemini başlatmak için &quot;Başlat&quot; a tıklayın. </li><li> Ablasyon lazer ateş önce, fare işaretçisi simgesi seçilir ve damar duvarında net bir odak olduğunu olduğundan emin olun. </li><li> Lazer atışı için çift damar duvarının 2-3 mikron dahili bir noktayı tıklayın. Arteriyollerde endotel hücrelerinin Yaralanma aydınlık alan görüntü fark olması gereken bir görsel bir şekil değiştirme olur, trombosit birikim izledi. Monitör trombüslazer yaralanmadan sonra 5 dakika boyunca oluşumu. </li><li> Lazer yaralanmadan sonra beş dakika bekleme (trombüs boyutu artık kararlı olmalıdır) ve yakalama iptal. Daha sonra, 20 dakika boyunca yapışkan trombositlere ve haddeleme / yapışık nötrofiller kaydetmek için 500 milisaniye aralığı ile 2400 kez puan ile bir yakalama başlar. Üç ila beş arteriyollerde bir fare kaydedilir. </li><li> Deneyin tamamlanmasından sonra, fareler servikal dislokasyon yolu ile ötenazi yapıldı. </li></ol> 6. Arteriolar Tromboz Model Veri Analizi <ol><li> 4,5 adımları 4.1 üzerine gidin. </li><li> Trombosit trombüs oluşumu sırasında arka plan floresan yoğunluğu elde etmek için, 4.14 adımlarda 4.7 üzerine gitmek. </li><li> &quot;Maske&quot; Git, &quot;Segment&quot;, kanal seçme FITC tıklatın ve Düşük arka plan sinyalin ortalama değerini ekleyin. Click &quot;Uygula&quot; ve &quot;Tamam&quot;. </li><li> Trombosit trombüs floresan yoğunluğu (Dylight 488-konjuge anti-CD42c antikor) analiz etmek, </strong&gt; 4.19 adımlarını 4.18 üzerine gitmek. Antikor sinyalini hesaplamak için, &quot;Sum Yoğunluğu&quot; (&quot;Maksimum Yoğunluk&quot; x &quot;Alan (piksel)&quot;) plan sinyali tarafından çıkarılır. Bu, trombosit trombüs floresans şiddetidir. </li><li> Haddeleme ve yapışık nötrofiller ölçmek için, timelapse oynamak ve gözle 20 dakika boyunca trombosit trombüs üzerinde rulo hücrelerinin sayısını saymak. En azından 2 saniye süre ile trombosit trombüs ile etkileşim sırasında Rolling nötrofil hızında bir azalma olarak tanımlanır. Bağlanan nötrofiller, en az 2 dakika süreyle trombosit trombus bağlı kalan herhangi bir nötrofiller olarak tanımlanır. Haddeleme ve yapışık nötrofil sayısı 20 Dakikada hücre sayıları olarak sunuldu. Rolling hızı bir partikül izleme sistemi kullanılarak hesaplanmıştır. </li></ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

Yazarlar hiçbir rakip finansal çıkara beyan.

Burada vasküler inflamasyon ve tromboz sırasında aktive endotel üzerindeki heterotipik trombosit-nötrofil etkileşimleri görselleştirmek için gerçek zamanlı floresan intravital mikroskopi için ayrıntılı bir protokol açıklar. Daha önce, benzer floresans mikroskopik yaklaşımlar trombüs oluşumu ve vasküler inflamasyonun moleküler mekanizması çalışmaya bildirildi. 8,12 heterotipik hücre-hücre etkileşimi yaralanma yerinde vazo-oklüzyon için önemli olabilir bu yana, bu teknoloji iç...

<table border="1">
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		<tr>
			<td>Name of Reagent/Material</td>
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		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
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			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

Arterioler yaralanma yerinde trombüs oluşumu sırasında aktive endotel aracılık tromboz ve vasküler inflamasyon üzerine aktive trombositler ve lökositler (özellikle nötrofiller) etkileşimi. 1,2, aktive endotel ve subendotelyal matriks proteinlerine yapışkan trombositlere nötrofil yuvarlanma ve yapışma destekliyoruz. 3 Tersine, venüler inflamatuar koşullarda, aktif endotele yapıştığında nötrofil trombositlerin dolaşımdaki adezyon ve birikimi destekleyebilir. Heterotipik trombosit-agregasyonu nötrofil hücreleri arasındaki spesifik reseptör-reseptör etkileşimleri yoluyla sayaç ardışık işlemler gerektirir. 4 O aktive edilmiş endotel hücreleri, böylece yüksek kesme koşulları altında trombosit yapışması ve birikim başlatılması, bu tür von Willebrand faktörü gibi yapışma molekülleri serbest olduğu bilinmektedir. 5 Ayrıca, aktive endotel hücreleri ifade selektinler nötrofil yuvarlanma ve adezyon bir destekd Hücrelerarası adezyon molekülü-1 (ICAM-1), sırasıyla, düşük kesme koşulları altında. 4 Trombosit P-selektin yoluyla nötrofiller ile etkileşime girer p-selektin glikoprotein ligandı-1 (PSGL-1), nötrofil ve böylece β2 integrinler ve sağlam aktivasyonunu uyararak İki hücre türleri arasındaki yapışma. Heterotipik trombosit-nötrofil etkileşimleri tam kan ya da izole edilmiş hücreler 6,7 olarak belirlendiği in vitro deneylerde gelişmelere rağmen, bu çalışmalar vasküler hastalık sırasında oksidatif stres koşulları manipüle edemez. Bu raporda, trombosit ve nötrofil işaretleyici bir fare karşı floresan-etiketli, spesifik antikorlar kullanılarak, TNF-α ile indüklenen inflamasyonu veya lazer kaynaklı takip sırasında aktive endotel üzerinde trombosit ve nötrofil heterotipik etkileşimlerini izlemek için ayrıntılı bir intravital mikroskopik protokol açıklar canlı farelerin cremaster kas mikrodamar yaralanma.

Ayrıntılı bir intravital mikroskopi analizi kullanarak, aktive endotel üzerindeki heterotipik trombosit-nötrofil etkileşimleri trombosit (CD42c) veya nötrofil işaretleyici (Gr-1) canlı farelere karşı floresan etiketli antikorlar infüzyonu ile görüntülenmiştir. TNF-α-kaynaklı venüler enflamasyon modelinde, en çok haddeleme nötrofiller stabil kayıt süresi (3-4,5 saat TNF-α enjeksiyonundan sonra, Şekil 2A&#39;da sırasında ICAM-1 ile aktive β2 İntegr...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Burada vasküler inflamasyon ve canlı farelerde trombüs oluşumu sırasında aktive endotel üzerindeki heterotipik trombosit-nötrofil etkileşimleri görselleştirmek için floresan intravital mikroskobu deneysel bir tekniktir rapor. Bu mikroskobik teknoloji vasküler hastalığın moleküler mekanizmasını incelemek ve patofizyolojik durumlardaki farmakolojik ajanları test için değerli olacaktır.

Canlı Fare Damar İltihabı ve trombüs oluşumu sırasında Activated Endotel üzerinde heterotypic Trombosit nötrofil Etkileşimleri Gerçek zamanlı Görüntüleme

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K Görüntüleme.  University of Illinois at Chicago. Burada vasküler inflamasyon ve canlı farelerde trombüs oluşumu sırasında aktive endotel üzerindeki heterotipik trombosit-nötrofil etkileşimleri görselleştirmek için floresan intravital mikroskobu deneysel bir tekniktir rapor. Bu mikroskobik teknoloji vasküler hastalığın moleküler mekanizmasını incelemek ve patofizyolojik durumlardaki farmakolojik ajanları test için değerli olacaktır.

Video: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Lökotrien B Gerçek zamanlı görüntüleme 4 Hücre aracılığı ile Göç ve β-arrestin BLT1 Etkileşimleri

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

İmmünoloji ve Enfeksiyon

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Vasküler Endotel Yüksek Lumen Basıncı Görüntüleme Lökosit Adezyon

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Normal MSS Real-time PCR ve Nöro sırasında mikroglia içinde microRNA tespiti

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T hücre gerçek zamanlı Canlı Görüntüleme Kompleks Oluşum Sinyal

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Nötrofil Hicret altında Fizyolojik Akış sırasında endotel hücre-hücre Eklemlerinin Gerçek zamanlı Görüntüleme

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; İnsan Tüm Kan ve Endotelyumunun Arasındaki Etkileşimler incelenmesi için Model

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Görüntüleme Patogenez ve Cording Deşifre ve<em&gt; Mycobacterium abscessus</em&gt; Zebra balığı Embriyolar

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Gerçek zamanlı Ekstrasellüler Flux Analizi Polarize M1 ve M2 Kemik İliği-türevli Makrofagların Metabolik Karakterizasyonu

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

In Vitro Mikroakışkan Hastalık Modeli Gerçek Zamanlı Olarak Tam Kan-Endotel Etkileşimleri ve Kan Pıhtısı Dinamikleri Çalışma

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...