This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

NOT: Hayvan prosedürleri İsviçre&#39;nin Cenevre şehrinde Hayvan Bakım kurumsal Etik Komitesi ve Kanton Veteriner Dairesi ile uygun olarak yapıldı. Yetkilendirme numarası GE / 63/14. Kemik iliğinden tek bir hücre süspansiyonu hazırlanması 1. <ol><li> Servikal dislokasyon ile Kurban fare (eski 8-12 hafta). % 70 etanol ile arka ayakları sterilize. Arka ayakları cildi çıkarın. </li><li> Fare uyluk ve kaval kemiklerinden inceleyin ve bir neşter ile bacaklarından doku kaldırmak. PBS ile dolu bir kültür çanak içine% 90 etanol ve yer ile bacakları durulayın. </li><li> Diz ekleminde bir neşter ve ayrı olan uyluk ve kaval kemiklerinden kültürü kaputu, temiz dokusu altında. Kemiklerin her bir ucunu kesti. </li><li> 50 ml&#39;lik bir 23G iğne ve hazırlama ortamı (fenol kırmızısız DMEM / F12,% 1 sıçan serum) ile dolu bir 1 ml şırınga kullanarak her bir kemikten kemik iliği yıkayın tüp vidalı. Ince 23G NE içinden Pasajlanması hücre agrega DisruptEdle ve 1 ml&#39;lik şırınga. Hazırlık ortamı ile, 25 ml toplam hacim getirin. </li><li> Santrifüj 250 xg 5 dak, 4 ° C. Atın süpernatant. 1 ml içinde süspanse pelet Eritrosit Lizis RBLC tamponu 15 ml tüp vidası (8.3 g NH4CI, distile su ile 1 L&#39;ye tamamlandı 1 g NaHCO 3, 1 ml EDTA (100 mM), 0,22 um filtre). Buz üzerinde 30 saniye inkübe edin. </li><li> Hazırlık orta ve santrifüj 250 x g&#39;de 10 ml, 5 dakika, 4 ° C ekleyin. 2 ml hazırlanmış sıvı supernatant ve tekrar süspansiyon atın. </li></ol> Negatif Seçim ile 2. Nötrofil Zenginleştirme <ol><li> Fare nötrofil zenginleştirme kokteyli (hücre izolasyonu platformu kiti gibi EasySep gibi) 150 ul ekleyin. Karıştırın ve 10 dakika boyunca buz üzerinde inkübe edilir. </li><li> Fenol kırmızısı içermeyen DMEM 10 ml ekleyin. Bir kez ters çevirin ve santrifüj 250 xg, 3 dakika, 4 ° C. </li><li> Atın süpernatant. Alt tüpler yuvarlak 5 ml polistiren 1.75 ml hazırlık ortamda süspanse pelet. 250 ul ekle BiotSeçim Kokteyl. Karıştırın ve 10 dakika boyunca buz üzerinde inkübe edilir. </li><li> Tanecikleri yeniden sübyeleştirmek için 30 saniye için (hücre izolasyon platformu kit) manyetik parçacıkları vorteksleyin. Hücre süspansiyonunun 500 ul manyetik parçacıkların ekleyin. Karıştırın ve 10 dakika boyunca buz üzerinde inkübe edilir. </li><li> Cazibe merkezi haline tüp yerleştirin. 3 dakika bekleyin. </li><li> İstenen Etiketsiz hücreleri toplamak için alt tüpler yuvarlak yeni bir 5 ml&#39;lik polistiren üzerine mıknatıs ters çevirin. Tüp içinde asılı son damlasına almayın. İstenmeyen hücre mıknatıs içine bir tüp içinde kalır. Bir biohazard atık içine bu tüp atın. </li><li> Mıknatıs yeni tüp yerleştirin. 3 dakika bekleyin. </li><li> Yeni 15 ml istenen etiketsiz hücreleri toplamak için tüp vidalı üzerine mıknatıs ters çevirin. Tüp içinde asılı son damlasına almayın. </li><li> Fenol kırmızısı serbest DMEM ile 5 ml Komple hacmi. </li></ol> Nötrofiller 3. Etiketleme <ol><li> Böyle Hücre Tracker Orange olarak turuncu bir boya 0.5 ul ekleyin (çalışma konsantrasyonu1 uM, CMRA - klorometil-RODOL-asetat, hazır DMSO içinde 10 mM). 37 ° C &#39;de 2 dakika kuluçkalayın. </li><li> Hazırlama ortamına 2.5 ml ilave edilir. Santrifüj 250 xg 5 dak, 4 ° C. </li><li> Atın süpernatant. 1.5 ml bir tüp içinde 1 ml hazırlanmış sıvı içinde süspanse pelet. </li><li> Hematositometre ile saymak için bir kısım atın. </li><li> 37 ° C&#39;de 5 dakika inkübe edin. Santrifüj 250 xg 5 dak, 4 ° C. </li><li> Atın süpernatant. 1 ml PBS içinde yeniden süspanse pelet. Santrifüj 250 xg, 5 dk, yıkamak için 4 ° C. </li><li> Atın süpernatant. 100 ul PBS başına 10x10 6 hücre olarak süspanse pelet. Iv enjeksiyon kadar buz üzerinde tutun. NOT: Hücreler iv mümkün olduğunca çabuk enjekte olması gerekir. 6-12x10 6 nötrofil / fare genellikle elde edilir. </li></ol> İntra vital mikroskopik 4. Fare Hazırlama NOT:.. Adım 4.1) ve 4.2) ic üzerinde etiketli nötrofil uzun bekleme süresini önlemek için denemenin başında yapılmalıdıre. <ol><li> CX 3 CR1 GFP fare (eski 8-12 hafta) tartılır. </li><li> (PBS içinde 1.75 mg / kg ketamin acepromazone 60 mg / kg, ksilazin 4,5 mg / kg), anestezik 800 ul hazırlayın. </li><li> Fare anestezisi. Fare için 200 ul / 20 g Enjeksiyon ip. Kısma ayak tarafından ve solunum hızını kontrol ederek Kontrol anestezi. </li><li> Nötrofiller (100 ul PBS içinde 10x10 6 hücre), venöz deneyci kolaylık, yanal kuyruk venleri veya retro-orbital sinüs enjekte edilir. </li><li> Isıtma yastığı fare koyun ve göz jeli ile gözleri korumak. </li><li> Bir cam lamel (35 mm çapında), bir 30 mm çapında bir delik olan bir 10 cm doku kültürü çanağı içinde konur. Kullanım yağı lamel ile temas halinde doku kültürü çanak tutmaktır. </li><li> Periton maruz makas ile karın cildi İnsizyon. Bağırsak maruz makas ile periton İnsizyon. </li><li> 200 ul PBS p haline (37 ° C&#39;de önceden ısıtılmış) ilaveeritoneal boşluğu. Elinizdeki fareyi alın ve bağırsak nazik bir basınç ile periton boşluğuna dışarı çıkar böylece, yüzü aşağı çevirin. Doku kültürü çanak fare yerleştirin. </li><li> Yavaşça mezenterik damarları açığa amacıyla lamel üzerine steril pamuk tomurcukları ile bağırsak tekrar turlama. </li><li> Bantlarında kağıt mendil kesin ve 37 ° C ısıtılmış PBS ile ıslatın. Peristaltizm kaynaklanan hareketleri azaltmak için kağıt dokuların adet bağırsak hareketsiz. </li><li> Ters mikroskop ısmarlama tepsi sahneye 37 ° C termostatlı hazne içerisindeki doku kültürü çanak ve fare koyun. Arka bacak içine anestezik sc içeren bir şırınga tanıtın. NOT: Ayrıca, fare bir maske ile oksijen (0.5 L / dk) alabilirsiniz. </li><li> Ayak kısma ve solunum hızı her 30 dakikada bir kontrol ederek anestezi Kontrol fare. Gerekirse, düzgün anesteziyi idame ettirmek için anestezik bir destek (20 ul) teslim. DEĞİLE: damar kurutulması kaçınılmalıdır. Bu nedenle, nem oranının düzenli 37 ° C ısıtılmış PBS ile bağırsak immobilize etmek için kullanılan kağıt mendil ıslatıcı tarafından yapılmaktadır. </li></ol> Intravital Mikroskobu kullanılarak In Vivo iltihaplı Gemiler 5. Ölçme Nötrofil ve Monosit Davranışı <ol><li> Set lazerler ve lazer 488- kaynaklı fototoksisite önlemek için gerekli olan en düşük güçte 561- etmek. Deneylerde, 0,5% &#39;den düşüktür. Nötrofillerinde monositlerinde GFP ve portakal boya Yoğunluğu genellikle% 0.3 yeterli olduğunu çok parlak olduğunu. NOT: Bu bizim sistemimizle 0.15 mW 0.25 bir lazer gücü temsil eder. </li><li> Ters mikroskop 20X kuru objektif kullanarak açık bir iğne deliği ile 2.2 saniyede 512 x 512 piksel (yani 635 mikron) görüntülerini elde edin. Düşük lazer güçler yeterli sinyal sağlayan 20 um 10 arasında bir z-derinlik kullanın. NOT: Biz genellikle 12 ila 16 arasında bir z-derinliği ile deneyler gerçekleştirmek56 m. Sunulan rakamlar ve film durumunda, z-derinlik 20 mm. Faz kontrast endotel duvar belirlenmesini sağlar ve görüntüleme mesenterik damarlar üzerinde gerçekleştirilir. Devriye gezen hücreler ilgi alanı kayıt, 5-10 mikron / dk 6 hızında oldukça yavaş hareket beri her 20 sn tatmin edicidir. Açıklanan ayarlar ile, motorlu sahne aynı anda ilgi kadar 7 alanların kayıt sağlar. </li><li> Onları ilk film edinimi önce 30 dakika boyunca hazırlık kurtarmak için izin vardır fare ve mikroskop termostat odasında mezenterik damarlar yerleştirin. Bu süre zarfında, ilgi alanlarını çeşitli seçin. Amacına en yakın geminin lümen yüzeyi üzerinde odaklanın. Gerekirse, odağı ayarlamak için endotel hafif otomatik floresan yararlanın. NOT: Fare hazırlanması sırasında Cerrahisi hafifçe endotelyumu vurguluyor. Sonuç olarak, nötrofillerin haddeleme ve/ veya monositler, ilk 15 dakika takiben fare hazırlanmasında gözlenebilir. </li><li> Kararlı hal şartları altında, 30 dakika, tek bir görüntü her 20 saniye için bir birinci film kaydedin. NOT: Yazılım 2.2 sn ilgi her alan kazanır ve motorlu sahne otomatik olarak diğer bir alandan geçer. Bu kayıt film 30 dakika boyunca geri 20 saniye içinde ilk pozisyona gider. </li><li> Kan damarı iltihabı başlatmak için, TLR2 100 ug içeren PBS 20 ul bir damla ekleme / TLR1 agonisti Pam3CSK4 12, doğrudan görüntü verilen gemilerin üzerine. </li><li> Ilgi her alan için odağı kontrol edin. NOT: edinim sırasında, her zaman vardır z ekseni üzerindeki odak küçük değişiklikler. 10-20 mikron z-derinliğine rağmen, bu floresans yoğunluğunu bir azalmaya neden olabilir. Gerekirse nedenle, el, her filmin sonunda ilgi her alan yönlendirmesi. 30 dakika-sürelerle ve 3 saat kadar Tutanak filmlerital inflamasyon başladıktan sonra. </li><li> Deneyin sonunda, servikal dislokasyon ile anestezi uygulanmış fare kurban. </li></ol> Film Dosyaları ve lökosit ve Takip 6. Nesil NOT: Nikon kazanım yazılım dosyalarını .nd2 üretir ancak aşağıdaki prosedür diğer yazılım ve markalar ile benzer olacaktır. <ol><li> Tiff serisi olarak ihracat dosyaları. </li><li> ImageJ yazılım (Git <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/).</a> Tiff serisi içe aktarın. Farklı bir dosya olarak Her kanal. </li><li> Arka plan gürültüsünü azaltmak için yeşil ve kırmızı kanallar (Süreç&gt; Filtreler&gt; Medyan ...) için 2.0 piksel yarıçaplı bir medyan filtre uygulayın. </li><li> (İkili olun&gt; Proses&gt; Binary) ikili dosyaları dönüştürün. Her görüntü için eşiği hesaplamak için yazılım etkinleştirin. </li><li> Bir resim serisi (Görüntü&gt; Renk&gt; ... Kanallar Merge) içine kanalları Birleştirme. Lenmesi, monositler seçinn kanal, kırmızı kanal ve gri kanalda iletilen ışık nötrofiller bulunmaktadır. </li><li> RGB renk içine yığılmış görüntüleri dönüştürün (Görüntü&gt; Renk&gt; RGB Stack). </li><li> Avi olarak dosyayı kaydedin </li><li> Veriler ithal ve Imaris yazılımı (Bitplane) derlenmiştir. Monosit ve nötrofillerin hareketleri sonra Nokta İzleme Sihirbazı kullanılarak izlenir. </li></ol>

<ol>
	<li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Nourshargh, S., Hordijk, P. L., Sixt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaching+multiple+barriers:+leukocyte+motility+through+venular+walls+and+the+interstitium.">Breaching multiple barriers: leukocyte motility through venular walls and the interstitium.</a> Nat Rev Mol Cell Biol. 11, 366-378 (2010).</li><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+migration+into+inflamed+tissues.">Leukocyte migration into inflamed tissues.</a> Immunity. 41, 694-707 (2014).</li><li>Ley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.+Intravital+microscopic+investigation+of+leukocyte+interactions+with+the+blood+vessel+wall.">Chapter 11. Intravital microscopic investigation of leukocyte interactions with the blood vessel wall.</a> Methods Enzymol. 445, 255-279 (2008).</li><li>Sperandio, M., Pickard, J., Unnikrishnan, S., Acton, S. T., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+leukocyte+rolling+in+vivo+and+in+vitro.">Analysis of leukocyte rolling in vivo and in vitro.</a> Methods Enzymol. 416, 346-371 (2006).</li><li>Auffray, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+blood+vessels+and+tissues+by+a+population+of+monocytes+with+patrolling+behavior.">Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior.</a> Science. 317, 666-670 (2007).</li><li>Cros, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD14dim+monocytes+patrol+and+sense+nucleic+acids+and+viruses+via+TLR7+and+TLR8+receptors.">Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors.</a> Immunity. 33, 375-386 (2010).</li><li>Hanna, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+NR4A1+(Nur77)+controls+bone+marrow+differentiation+and+the+survival+of+Ly6C-+monocytes.">The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes.</a> Nat Immunol. 12, 778-785 (2011).</li><li>Woodfin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junctional+adhesion+molecule+JAM-C+regulates+polarized+transendothelial+migration+of+neutrophils+in+vivo.">The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.</a> Nat Immunol. 12, 761-769 (2011).</li><li>Proebstl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+support+neutrophil+subendothelial+cell+crawling+and+breaching+of+venular+walls+in+vivo.">Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo.</a> J Exp Med. 209, 1219-1234 (2012).</li><li>Wantha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-derived+cathelicidin+promotes+adhesion+of+classical+monocytes.">Neutrophil-derived cathelicidin promotes adhesion of classical monocytes.</a> Circ Res. 112, 792-801 (2013).</li><li>Ozinsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repertoire+for+pattern+recognition+of+pathogens+by+the+innate+immune+system+is+defined+by+cooperation+between+toll-like+receptors.">The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.</a> Proc Natl Acad Sci U.S.A. 97, 13766-13771 (2000).</li><li>Lammermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+swarms+require+LTB4+and+integrins+at+sites+of+cell+death+in+vivo.">Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo.</a> Nature. 498, 371-375 (2013).</li><li>Carlin, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nr4a1-dependent+Ly6C(low)+monocytes+monitor+endothelial+cells+and+orchestrate+their+disposal.">Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.</a> Cell. 153, 362-375 (2013).</li><li>Hamon, P., Rodero, M. P., Combadiere, C., Boissonnas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+mouse+bone+marrow+monocytes+in+vivo.">Tracking mouse bone marrow monocytes in vivo.</a> J Vis Exp. , (2015).</li><li>Iqbal, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD68+promoter+GFP+transgenic+mice+allow+analysis+of+monocyte+to+macrophage+differentiation+in+vivo.">Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.</a> Blood. 124, e33-e44 (2014).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PloS One. 6, e25109 (2011).</li></ol>

The authors declare that they have no competing financial interests.

Bu yazıda anlatılan metodolojiler verimli kararlı durum ve inflamatuar koşullarda mezenterik venlerde monosit ve nötrofil davranışlarını incelemek için tutarlı bir yaklaşım sağlamak. Hazırlık önemli adım PBS ıslak mendil ile bağırsak immobilizasyonu olduğunu. Düzgün gerçekleştirilen Eğer mezenterik damarlar güzel görüntü elde etmek için lamel maruz kalmaktadır. Bu mezenterik venlerde lökosit davranışını izlemek için ilgi çeşitli alanlarda seçimini sa...

<html> Nötrofiller ve monositler sürekli dolaşıma doğuştan gelen bağışıklık sistemi hücreleri bulunmaktadır. Yaralanma ya da enfeksiyon üzerine, enflamatuar sinyaller burada hücresel bir bağışıklık tepkisi başlatılması 1-3, hasar görmüş enfekte dokulara lökosit ROS neden olur. Lökosit seferberlik hızlılığı bağışıklık tepkilerinin olumlu sonucunu belirler. Bu karmaşık süreçler lökosit karışmış moleküller üzerinde anlayışlar elde .To dolaşan lökositler ve endotelyum 1-3 arasındaki yapışma temasların kurulması için yardım iltihaplı endotel üzerinde bulunan belirli moleküllerin (örneğin, selektinler, endotel bağlı kemokinler) güveniyor işe alım çağlayan, hücre işe kinetiği görselleştirmek için ve her bir hücre / nüfus davranışlarını izlemek için önemlidir. Etkili bir yöntem ilgi hücrelerinde floresan proteinleri eksprese eden transgenik fareler üzerinde İntra vital mikroskopik içerir. Bugüne kadar içeriği &quot;&gt;, intravital mikroskopi kullanılarak çeşitli yaklaşımlar görüntüye geliştirilen damar 4,5. Mesela, kulak dermis damar sistemi ya da intravital konfokal mikroskobu ile mezenterik damarların görüntüleme kemirgen Ly6C düşük monosit ve insan devriye davranışını tanımlamak için kullanılan CD14 kararlı hal şartları altında 6-8 kan damarlarının lümen tarafında CD16 + monositleri dim. Fare cremaster damar modeli, genellikle transgenik farelerin enflamatuvar veya iskemik koşullar altında nötrofil veya enflamatuar Ly6C yüksek monosit davranışını izlemek için kullanılır. Bu ise durumda, Cremaster IL1β, CCL2, TNFβ ya fMLP intraskrotal enjeksiyon ile uyarılır. 2-4 saat sonra, dokular daha sonra cerrahi dışarı çıkarılmıştır ve intravital konfokal mikroskopi 9-11 ile analiz edilmiştir. Aşağıdaki protokol bir ile aynı anda monositleri ve nötrofilleri izlemek için bir yöntemi tarif etmektedirters floresan konfokal mikroskop. Ayrıca, yöntem lökosit işe kinetiği takip nasıl daha önce görüntü aynı geminin (kararlı hal durumu) ve inflamasyon sonra nasıl ayrıntıları. Bu amaç için, olan monositler eGFP ifade iv portakal renkli bir boya etiketli kemirgen nötrofillerinin, enjekte CX 3 CR1 gfp / ağ fare kullanılır. Ters konfokal mikroskop kullanılarak, (1) izler ve kararlı koşullar ve (2) lokal enflamasyon sonrasında aynı kap içinde iki monosit ve nötrofillerin ilerlemesini takip etmek altında devriye Ly6C düşük monositleri analiz etmek mümkündür. Burada TLR2 / TLR1 agonist Pam3CSK4 12 kullanarak inflamatuar koşullar yaratır. Ayrıca, bu tür görüntüleme belirli nakavt fareler üzerinde ya da engelleme antikorların 6,9,13 varlığında gerçekleştirilir ise lökosit işe çağlayan çeşitli adımlar ilgi belirli moleküllerin rolünü belirlemek için yardımcı olabilir.

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

Etkin bağışıklık tepkisi enfeksiyon veya hasar bölgesinde kan lökositlerinin hızla harekete bağlıdır. In vivo lökosit göçü incelenmesi damar endotel ile lökosit endotel göç ve etkileşim moleküler temelini anlamak için çok önemlidir. Bir etkili bir yaklaşım ilgi hücrelerinde floresan proteinleri eksprese eden transgenik fareler üzerinde İntra vital mikroskopik içerir. Burada bir ters konfokal mikroskop ile turuncu boya etiketli nötrofil enjekte CX 3 CR1 GFP / ağırlık fare iv görüntüleme monosit ve nötrofillerin için bir protokol mevcut. Time-lapse film hem kararlı hal ve inflamatuar koşullarda mezenterik venlerde lökosit davranışının analizine imkan görüntüleme birkaç saat 30 dk toplandı. Yerel TLR2 / TLR1 agonisti Pam3SK4 kan damarı inflamasyonunu indüklemek ve subseque izlemek Ayrıca adımları açıklarnötrofil ve monositlerin nt işe. Sunulan teknik aynı zamanda lökositlerin diğer popülasyonları izlemek ve diğer uyaranlara veya transgenik fareler kullanılarak lökosit alımı veya insan ticareti karışmış molekülleri araştırmak için kullanılabilir.

Eser gerçek zamanlı olarak kolayca anestezi farelerin mesenterik damarlar içindeki monosit ve nötrofillerin davranışını izlemek için optimize edilmiş bir protokol açıklamaktadır. 37 ° C-termostatlı odacığın kullanımı fare sıcaklığı korumak için zorunludur ve aynı zamanda nedeniyle lökositlerin sıcaklığa bağlı hareket etmektir. Fare hazırlanması Şekil görüntülenir 1. Şekil 2 mikroskop altında görülen tüm bölgeyi gösterir. İletilen ışık mez...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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 ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K Görüntüleme.  University of Geneva. 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.

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

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

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

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

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

İmmünoloji ve Enfeksiyon

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

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

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 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Etkileşimi incelenmesi<em&gt; Staphylococcus aureus</em&gt; Akım Sitometri ve Time Lapse Mikroskopi tarafından Nötrofiller ile

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

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, ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Cevabı İnsan İlköğretim nötrofiller ve monositler tarafından Antifungal Faaliyet Canlı Görüntüleme<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

İki fotonlu Intravital mikroskobu kullanılarak grip virüsü enfeksiyonu sırasında trakeal mukozasında görüntüleme hücre etkileşimi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

İntravital Mikroskopi Ile Değerlendirilen Farelerde Cremaster Kasın Lökosit İnfiltrasyonu

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...