We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nad&#232;ge Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales" (ANRS) including a doctoral fellowship for JM.

Not: LifeAct mCherry kullanılan plazmid 17 sonra üretilen Dr. Guillaume Montagnac, Institut Curie, Paris, bir tür hediyedir. 1. Hücre ve transfeksiyonu Not: RAW264.7 makrofaj komple ortam içinde, alt konfluent (RPMI (Roswell Park Memorial Institute) 1640 ortamı, 10 mM HEPES, 1 mM sodyum piruvat, 50 uM β-mersaptoetanol, kadar büyütülür, 2 mM L-glutamin ve% 10 FCS ( 100 mm plaka fetal inek serumu)). Bunlar elektroporasyon ile floresan etiketli proteinleri kodlayan plazmid ile transfekte edilir. Rutin olarak, yaklaşık 5-6 Ekim 6 hücreleri, sırasıyla, her transfeksiyon ya da ko-transfeksiyon için plazmid, 20 ug veya 10 ug ile transfekte edilir, x. Elektroporasyon veya lipofeksiyon göre transfeksiyon için diğer araçlar alternatif yaklaşımlar olarak kullanılabilir unutmayın. <ol><li> Hücre kaldırıcı hücreleri kazıyın ve pipetleme ve birkaç kez aşağı kültür ortamında 10 ml bunları tekrar süspansiyon. </li><li> santrifüjHücre süspansiyonu 300 xg, sallanan açılı rotor oda sıcaklığında 5 dakika. </li><li> tam orta Onceden 10 mi, 37 ° C de gentamisin 10 ug / ml ile takviye edilmiştir. </li><li> Santrifüj sonrasında, süpernatant atılır ve elektroporasyon kitinden &quot;yıkama tamponu A&quot; 3 ml pelletini. </li><li> hücre süspansiyonu 300 xg, sallanan açılı rotor oda sıcaklığında 5 dakika santrifüj. </li><li> Bu süre içinde, oda sıcaklığında, DNA karışımı hazırlamak: 120 ul 2x tampon B, ilgilenilen proteinin, QSP 240 ul H2O kodlayan plazmit DNA 20 ug </li><li> santrifüj sonrasında, tüm süpernatant atın. </li><li> DNA karışımı hücre pelletini ve 4 mm elektroporasyon küvetler içine aktarın. </li><li> 3 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 250 V, 900 iF de electroporate. </li><li> Hemen gentamisin ile tamamlanmış olan önceden ısıtılmış (37 ° C) &#39;de hücreler komple ortam tekrar süspansiyon ve plaka100 mm tabak m. </li><li> Gece boyunca 37 ° C,% 5 CO2 de transfekte edilmiş hücrelerin inkübe edin. </li><li> Ertesi sabah, (fenol kırmızısı olmaksızın RPMI 1640, 10 mM HEPES, 1 mM sodyum piruvat, 50 uM β-mersaptoetanol, 2 mM L-glutamin) serumsuz mikroskopi ortamı 10 ml tam orta yerine. </li></ol> Kırmızı kan hücrelerinin 2. Opsonizasyon Not: makrofajlar parçacık hedef bir model olarak, koyun kırmızı kan hücreleri (SRBC&#39;ler) kullanılır. Genellikle, 35 mm cam alt tabak başına yaklaşık 7 x 10 6 SRBC&#39;ler kullanılır. <ol><li> 1x PBS (fosfat tamponlu tuzlu su) /0.1% BSA (sığır serum albümini) ve santrifüj (600 x g&#39;de, 4 dakika), 100 ul SRBClere yıkayın. </li><li> Santrifüj sonrasında, süpernatant atılır ve 1 x 100 ul PBS /% 0.1 BSA ve santrifüj (600 x g&#39;de, 4 dk) SRBClere tekrar süspansiyon. </li><li> santrifüj sonrasında, süpernatantı atmak ve tavşan IgG anti-SRBClere 1x PBS /% 0.1 BSA SRBClere tekrar süspansiyonkonsantrasyonun alt-aglütinasyon. SRBClere antikor / 5 ul ihtiva eden çözeltinin 500 ul kullanın. Not: antikorların alt aglütinasyon konsantrasyonu ağ oluşumu olduğu tespit aglütinasyon uyarmadı düşük konsantrasyona karşılık gelir. Genel olarak, alt aglütinasyon konsantrasyonu 8.2 ug / ml&#39;dir. <ol><li> Bir hemaglütinasyon testi ile SRBClere opsonize için gerekli IgG anti-SRBClere konsantrasyonunu belirlemek. Bir mikro-1 / 25,600 - seri olarak 1/50 ile (13.1 mg / ml stok) IgG SRBC&#39;ler seyreltin. Her kuyuda 2 x 10 6 SRBClere ekleyin. Birkaç saat oda sıcaklığında karanlıkta plaka inkübe edin. </li></ol></li><li> yavaş dönüşü 30 dakika boyunca oda sıcaklığında inkübe edin. </li><li> 1x PBS içinde iki yıkama / yukarıda tarif edildiği gibi% 0.1 BSA sonra önceden ısıtılmış serum barındırmayan ortam içinde mikroskopi IgG opsonize SRBClere (IgG SRBC&#39;ler) (2 mi / çanak) yeniden süspanse edin. </li></ol> Lameller 3. Poli-lizin Kaplama <ol><li> Bu aradaOda sıcaklığında 30 dakika boyunca% 0.01 poli-L-lizin, 2 ml 35 mm cam alt yemekler tedavi. </li><li> 1x 2 ml PBS yemeklerin iki kez yıkayın. </li></ol> Cam alt yemekleri üzerine SRBClere 4. Kovalent olmayan Fixation <ol><li> 35 mm cam alt çanak başına SRBC&#39;ler süspansiyon 2 ml dökün. </li><li> 35 mm cam alt yemekleri üzerine 2 dakika boyunca 500 xg&#39;de bir sallanan rotor santrifüj. </li><li> Süpernatantı ve 1 x PBS /% 10 BSA, 2 ml ile bir kez yıkanır. </li><li> tabak başına 1 x PBS /% 10 BSA, 2 ml 30 dakika boyunca parçacıkları inkübe edin. </li><li> 1x 2 ml PBS yemeklerin üç kez yıkayın. </li><li> Önceden ısıtılmış (37 ° C), serumsuz mikroskopi orta 2 ml 1 x PBS değiştirin. </li></ol> 5. Fagositoz TIRFM tarafından görüntülendi <ol><li> Mikroskop Not: TIRFM bir yağ batırma objektifi (K 100 X, NA1.49), CO2 ile bir ısıtma odası ile donatılmış bir mikroskop kullanılarak gerçekleştirilmiştir ve iki şarkıBir 1.5x lens ile birleştiğinde le foton algılama kameraları EMCCD (Elektron Çarpma Charge Coupled Device). <ol><li> TIRF mikroskop oturumu önceki gün, mikroskop sahne homojen bir ısıtma sağlamak için 37 ° C&#39;de ısıtma odasına açın. </li></ol></li><li> Kritik Açı tayini Not: ImageJ Renk Profiler yazılım TIRF görüntü akışlarını işlemek için kullanılır. <ol><li> mikroskop altında serum içermeyen mikroskobu ortamıyla opsonize SRBC içeren bir 35 mm cam alt çanak yerleştirin. </li><li> Hücreleri Pençe ve eklemeden önce orta onları tekrar süspansiyon - çanak (100 500 ul). </li><li> mikroskop kontrol etmek ve 491 nm ve / veya floresan etiketli proteinleri ifade eden hücreleri belirlemek için bir 561 nm lazer ile uyarma yapmak için &quot;canlı Toplama&quot; yazılımı kullanın. </li><li> floresan etiketli proteinleri ifade eden bir hücreyi tespit. </li><li> alanın ortasına yerleştirin ve 500 im kazanmakFarklı açılarda bir uyarma dalga boyunda yaşları 0.01º (Şekil 2A) bir artış ile, 5º&#39;lik kadar 0º itibaren. açıları otomatik mikroskop sistemi tarafından değiştirilir. </li><li> olay ışık tamamen cam / orta arayüzü yansıyan sağlayan kritik açısını belirlemek ve kaybolan dalga üretir. ImageJ Renk Profiler yazılımı kullanarak, &quot;Dosya&quot; tıklayarak &quot;Aç&quot; görüntü sırası açın ve dosyayı seçin. </li><li> Düzgün bir floresan (Şekil 2B sarı 1) ile hücrede, dikdörtgen aracı ile, faiz (ROI) bir bölge seçin. </li><li> &quot;Z ekseni profili&quot; arsa (menü ve &quot;Plot Z ekseni Profili&quot; açılır &quot;Resim&quot; sekmesinde &quot;Stacks&quot; Şekil 2B tıklayarak x ekseni üzerindeki açıların fonksiyonu ile ROI ölçülen floresan yoğunluğu ortalama kırmızı 2). Not: Kritik açı ma giden açısıdırfloresan keskin bir düşüşe önce ximum floresan. </li><li> Örnek 2.00 (Şekil 2C) gibi bir TIRF sinyali elde etmek mikroskopi oturumu sırasında kritik açıya üstün x ekseni üzerindeki açısının herhangi bir değer kullanın. </li></ol></li><li> Devralmalar <ol><li> Canlı Toplama yazılımı kullanarak, modül &quot;Protokol Editor&quot; (Şekil 3A) ile iktisap parametrelerini ayarlamak. <ol><li> TIRF bölgedeki ilgi proteinlerin floresan sinyal elde etmek &quot;Çok Kanallar&quot; edinimi oluşan bir &quot;döngü&quot; ile bir protokol oluşturun. (Örneğin 2.00) TIRF açısı, (Örnek 50 ms için) kalma süresi ve (örneğin,% 50) lazer yoğunluğu (Şekil 3B 1) girin. </li><li> &quot;Çok Kanallar&quot; aracı ile Epifloresans sinyal edinimi elde etmek için TIRF bölgenin üstüne hedefi 3 mikron &quot;Z hareket&quot; tanıtmak. t altında bir açı girinO kritik açı (örneğin 1.00 gibi), fuar süresi (50 msn) ve lazer yoğunluğu (% 50) (Şekil 3B, 2 ve 3). </li><li> Sonraki TIRF bölge (Şekil 3B 4) dönmek için, aşağıda amaç 3 mikron &quot;z hareket&quot; ekleyin. (Işık Yayan Diyot) (Şekil 3B 5) LED parlak ışıkta hücrenin bir &quot;anlık&quot; ekleyin. </li></ol></li><li> parçacık etrafında plazma zarı uzatarak SRBC fagositoz başlatır floresan takılı protein ifadesi orta seviyede ilgi bir hücreyi bulun. </li><li> alanın ortasına yerleştirin. </li><li> 1000 kare - 500 edinilmesini akışı başlar. &quot;Döngü sayısı&quot; sekmesinde, &quot;750 kare&quot; (Şekil 3B 1) girin. Not: ImageJ Renk Profiler yazılım TIRF görüntü akışlarını işlemek için kullanılır. </li><li> tıklayarak Resim J yazılımında dizi görüntüleri açın&quot;Aç&quot; &quot;Dosya&quot; ve dosyayı seçerek. </li><li> sekme &quot;Image&quot; butonuna tıklayarak kanalları ayırmak, &quot;Hyperstacks&quot; menüsü ve &quot;Yığın hyperstack&quot; fonksiyonu açılır. çıktı penceresini tamamlayın: Sipariş: xyctz; Kanal (C): kanal sayısı (örn: &quot;2&quot;, iki fluorochromes varsa); Dilimler (z): z ekseninde dilim sayısı (örn: &quot;1&quot; z ekseninde hiçbir hareket yoksa); Çerçeveler (t): kanal sayısı başına bölünmüş görüntü sayısı; Ekran modu: Gri Tonlama. Not: İki ayrı görüntü dizileri iki farklı kanallar karşılık, oluşturulur. </li></ol></li></ol>

<ol>
	<li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Ann Rev Pathol. 7, 61-98 (2012).</li><li>Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+cups+into+phagosomes+and+macropinosomes.">Shaping cups into phagosomes and macropinosomes.</a> Nat Rev Mol Cell Biol. 9, 639-649 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+comparative+innate+immunity:+learning+on+the+fly.">Phagocytosis and comparative innate immunity: learning on the fly.</a> Nat Rev Immunol. 8, 131-141 (2008).</li><li>Niedergang, F., Bradshaw, R. A., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cell Biology. 2, 751-757 (2016).</li><li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+clearance:+basic+biology+and+therapeutic+potential.">Apoptotic cell clearance: basic biology and therapeutic potential.</a> Nat Rev Immunol. 14, 166-180 (2014).</li><li>Aderem, A., Underhill, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+phagocytosis+in+macrophages.">Mechanisms of phagocytosis in macrophages.</a> Annu. Rev. Immunol. 17, 593-623 (1999).</li><li>Underhill, D. M., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+during+phagocytosis.">Information processing during phagocytosis.</a> Nat Rev Immunol. 12, 492-502 (2012).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu Rev Immunol. 20, 825-852 (2002).</li><li>Canton, J., Neculai, D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenger+receptors+in+homeostasis+and+immunity.">Scavenger receptors in homeostasis and immunity.</a> Nat Rev Immunol. 13, 621-634 (2013).</li><li>Freeman, S. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+receptors,+signal+integration,+and+the+cytoskeleton.">Phagocytosis: receptors, signal integration, and the cytoskeleton.</a> Immunol Rev. 262, 193-215 (2014).</li><li>Scott, C. 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=Phosphatidylinositol-4,5-bisphosphate+hydrolysis+directs+actin+remodeling+during+phagocytosis.">Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.</a> J Cell Biol. 169, 139-149 (2005).</li><li>Schlam, 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=Phosphoinositide+3-kinase+enables+phagocytosis+of+large+particles+by+terminating+actin+assembly+through+Rac/Cdc42+GTPase-activating+proteins.">Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.</a> Nat Commun. 6, 8623 (2015).</li><li>Marion, 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=The+NF-kappaB+Signaling+Protein+Bcl10+Regulates+Actin+Dynamics+by+Controlling+AP1+and+OCRL-Bearing+Vesicles.">The NF-kappaB Signaling Protein Bcl10 Regulates Actin Dynamics by Controlling AP1 and OCRL-Bearing Vesicles.</a> Dev Cell. 23, 954-967 (2012).</li><li>Loovers, H. 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=Regulation+of+phagocytosis+in+Dictyostelium+by+the+inositol+5-phosphatase+OCRL+homolog+Dd5P4.">Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4.</a> Traffic. 8, 618-628 (2007).</li><li>Deschamps, C., Echard, A., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+cytokinesis:+do+cells+use+common+tools+to+cut+and+to+eat?+Highlights+on+common+themes+and+differences.">Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences.</a> Traffic. 14, 355-364 (2013).</li><li>Johnson, D. S., Jaiswal, J. K., Simon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12,+Unit+12.29:+Total+internal+reflection+fluorescence+(TIRF)+microscopy+illuminator+for+improved+imaging+of+cell+surface+events.">Chapter 12, Unit 12.29: Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.</a> Curr Protoc Cytom. , (2012).</li><li>Riedl, 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Marie-Anais, F., Mazzolini, J., Herit, F., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-actin+cross-talk+contributes+to+phagosome+formation+and+closure.">Dynamin-actin cross-talk contributes to phagosome formation and closure.</a> Traffic. 17 (5), 487-499 (2016).</li></ol>

The authors have nothing to disclose.

The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 &#176;C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We notice...

Fagositoz tanıma ve yutulur malzemenin içselleştirme ve bozulmasına yol açar, reseptörleri yüzey malzemesinin bağlanmasını başlar başlıca bir hücre fonksiyonudur. Böyle kalıp Dictyostelium discoideum ve amip gibi tek hücreli ökaryotlar bakteriler üzerinde beslemek için fagositoz kullanırken, yüksek organizmalar profesyonel hücreleri ile gelişmiştir. Makrofajlar ya da dendritik hücreler, çeşitli doku ve organlarda patojenlere karşı ilk savunma hattıdır ve antijen sunumu ve sitokin üretimi 1-4 ile adaptif bağışıklık sistemini aktive etmek için çok önemlidir. Belirli koşullar altında, fagositoz profesyonel olmayan fagositik hücreler, örneğin, endotel ve epitel hücreleri tarafından gerçekleştirilebilir. Bu işlem gelişimi sırasında ve normal doku devirinde ve yeniden modelleme için erişkin homeostazın muhafaza etmek önemlidir. Böyle testiste veya retina Sertoli hücreleri Son olarak, özel fagositlerpigment epitel hücreleri son derece güçlü fagositler 5 vardır. Bir fagozomun oluşumu burada mikroorganizmalar veya hücresel döküntü bozunması fagositik hücrenin yüzeyi üzerinde fagositik reseptörlerinin kümelenme ile başlar oluşur. sistemlerinde, Fc alıcıları (FcR) ya da tamamlayıcı reseptör (CR) ve opsonik reseptörlerin kümeleme, aşağıdaki akıntı yönündeki sinyal olaylarını de karakterize edilmiştir. Ancak, Toll benzeri reseptörler (TLR), lektinler, mannoz reseptörleri ve çöpçü reseptörleri de dahil olmak üzere pek çok sivil opsonik reseptörleri de vardır. Bu reseptörler, mannoz veya fukoz kalıntılarının, fosfatidilserin ve lipopolisakkaritler 1,6-9 parçacık yüzeyi üzerinde determinantlar tanır. Patojen veya hücre enkaz tanıma ve daha sonra yoğun ve geçici aktin yeniden yol fagositik reseptör çeşitli türleri, kümeleme bağlayıcı içerir. hücre içi compartmen paralel fokal ekzositozdats membran geriliminin serbest bırakılmasından katkıda bulunur ve büyük partiküllerin etkin fagositoz önemlidir. aktin polimerizasyonu ve membran deformasyon yol açan sinyal olayları tek bir fagositik reseptör tetikleyen deneysel modellerde disseke edildi. FcR aracılı fagositoz sırasında küçük GTPases (rac, Cdc42) tarafından düzenlenir yoğun bir aktin polimerizasyon bulunmaktadır. Bunların efektörlere arasında, Wiskott-Aldrich sendromu proteini (WASP) aktin filamanlarını 1,2,4,10 çekirdeklerin Aktin-ilişkili protein kompleksi 2/3 (ARP2 / 3) aktivasyonuna yol açar. Fosfatidilinositol-4, 5-bifosfat (PI (4,5) P2) Yerel üretim pseudopod oluşumunu sürücüler ilk aktin polimerizasyonu için çok önemlidir. PI (3,4,5) P3 olan dönüşüm pseudopod uzatma ve fagosom kapak 11 için gereklidir. Çeşitli yollar PI (4,5) P 2 kayboluşu katkıda bulunur. fosfatidilinositol fosfat kina öncelikle dekolmanıfagosom tutukladı PI (4,5) P2 sentezi gelen ses (PIPKIs). İkinci olarak, fosforile edilebilir ve ben kinazlar (PI3K) PI3K sınıf tarafından tüketilen ve PI (3,4,5) P3 12 dönüştürülür. Fosfatazlar ve fosfolipaz için bir rolü, memeli hücrelerinde ve Dictyostelium 13,14 fagositoz sırasında PI (4,5) P2, hidroliz ve F-aktin çıkarılması ima edilmiştir. Fosfolipaz C (PLC) δ diasilgliserol ve inositol-1,4,5- tris fosfat PI (4,5), p, 2 hidrolize olur. PI (4,5) P2 ve PI (3,4,5) P3 fosfataz OCRL (Lowe oculocerebrorenal sendromu), aynı zamanda fagosom oluşumunda rol oynadığı gösterilmiştir. F-aktin ve depolimerizasyon hassas yerel oluşumu sıkıca uzayda ve zamanda düzenlenir ve biz hücre içi bölmelerin o işe göstermiştir böylece fagositik fincan dibinde yerel aktin depolymerization katkıda yerel OCRL fosfataz teslim etmek önemlidir <sup&gt; 13,15. Bunun için, biz burada açıklanan deney seti kadar kullanılır. fagosom kapatılması ve membran açılmasıyla meydana için gerekli mekanizma ve moleküler oyuncular kötü çünkü görselleştirme ve fagosom kapatılması siteyi takip zorluklar tanımlı kalır. Yakın zamana kadar, fagositoz zor fagosom kapatma sitenin zamanında görselleştirme yaparak, onların sırt yüzünde ya da iki tarafta parçacıkları içselleştirmek sabit veya canlı hücreler gözlendi. Buna ek olarak, sabitleme yöntemleri membranların ve önyargı yalancı ayak uzatma ve kapatma sonuçları geri çekilmesini neden olabilir. Buna karşılık, biz kurmak ve burada açıklamak var tahlil bize pseudopod uzantısı ve toplam iç yansıma mikroskobu (TIRFM) 16 dayalı canlı hücrelerde 13 fagositoz kapatılması adımını, görüntülemenizi sağlar. Bu optik bir teknik şeffaf arasındaki ara yüzeyde ince bir bölgede florofor uyandırmak üzere bir çabuk kaybolan dalga harcayacaktırkapak (lamel) ve bir sıvı (hücre kültür ortamı). uyarım derinliği kalınlığı plazma zarına yakın bir moleküler olayların canlandırılmasına olanak tanıyan bir katı yüzey yaklaşık 100 nm&#39;dir. TIRFM yüksek sinyal-zemin oranı sağlar ve bağlı hücrelerin aydınlatma toplanan out-of-focus floresan ve sitotoksisite sınırlar. TIRFM yararlanan biz lamelleri IgG opsonize kırmızı kan hücrelerinin (IgG SRBC&#39;ler) ile kaplanmış sonra poli-lizin ile aktive edildiği &quot;fagosom kapatma tahlil&quot; geliştirdi. ilgi geçici floresan etiketli proteinler ifade makrofajlar daha sonra IgG SRBClere yutmak için izin verilir. Hücreler kovalent olmayan cam yüzeyine bağlanmış olan hedef partikülleri ayırmak birlikte, psödopod uçları görülen ve TIRF modunda kaydedilebilir. TIRF satın almalar vis sağlayan, yukarıdaki aşama 3 mikron değişen sonra, Epifloresans modunda satın almalar ile birleştirilirfagositik fincan baz ualization. Bu işlem sırasında, aktin ya da Dynamin inhibe gibi farmakolojik ilaçların ilavesi, moleküler seviyede işlemini incelemek için de mümkündür. Burada detaylı bir şekilde tarif protokol RAW264.7 mürin makrofaj hücre çizgisinde ve opsonize edilmiş parçacıklar için olduğu ancak, herhangi bir diğer fagositik bir hücre ve boncuk gibi başka hedeflere sahip uyarlanabilir. Bu yöntem zaman ve çeşitli fagositik sürecinde pseudopod uzantısı ve fagosom kapatılması rol oynayan moleküler oyuncuların uzayda düzenlemenin daha iyi karakterizasyonu sağlayacaktır.

<table><tbody><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td></td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cuvettes 4 mm</td><td>Cell project</td><td>EP104</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>Room temperature</td></tr><tr><td>Electrobuffer kit</td><td>Cell project</td><td>EB110</td><td></td></tr><tr><td>100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Gene X-cell pulser</td><td>Biorad</td><td>165-2661</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1397</td><td></td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td></td></tr><tr><td>iMIC</td><td>TILL Photonics</td><td></td><td>Oil-immersion objective (N 100X, NA1.49), heating chamber with CO&lt;sub&gt;2&lt;/sub&gt;, a camera single photon detection EMCCD (Electron Multiplying Charge Coupled Device) and a 1.5X lens</td></tr><tr><td>Poly-L-Lysine Solution 0.1%</td><td>Sigma</td><td>P8920-100ml</td><td>Dilution at 0.01% in water&amp;nbsp;</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td></td></tr><tr><td>RPMI 1640 medium, no phenol red (10 x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr></tbody></table>

"phagosome closure assay" to visualize phagosome formation in three dimensions using total internal reflection fluorescent microscopy (tirfm)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

Bu yazıda tarif edilen deneysel sistem şematik olarak Şekil 1 &#39;de gösterilmiştir. Bir floresan etiketi kaynaşık yararlı proteinleri eksprese eden transfekte RAW264.7 makrofaj kovalent olmayan bir şekilde tespit edildi IgG opsonize koyun kırmızı kan hücreleri (SRBC&#39;ler) ile temas içine yerleştirilmektedir lamel. makrofajlar onu yutmak için lamel SRBC ayırabilirsiniz. kullanılan TIRF mikroskop 3 mikron yukarıda Z vardiyadan sonra Epifloresans mod...

Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagozomun Kapatma Deneyi&quot; kullanma Üç Boyutta fagozomun Formasyonu Visualize&#39;a Toplam İç Yansıma Floresan Mikroskobu (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

phagosome-closure-assay-to-visualize-phagosome-formation-three

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

6.4K Görüntüleme.  Université Paris Descartes, Sorbonne Paris Cité. We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Video: "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

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

İmmünoloji ve Enfeksiyon

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Canlı Makrofagların içinde Phagolysosome Biogenesis&#39;den Çalışması

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Makrofaj Fagositoz ve Aktin Polimerizasyon Değerlendirme Yüksek Verimli Florimetrik Tekniği

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Oranlı metrik Floresan Mikroskopi fagosom pH ölçümü

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Nötrofil fagozomun Görüntüleme ve Sitoplazma bir oranlı metrik pH Göstergesi kullanma

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Fagositoz fare makrofajlar tarafından hızlandırılmış 3D görüntüleme

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF Mikroskopi ile Tek olay ebatları da G protein-bağlanmış alıcılar üzerinden Klatrin dolayımlı endositoz görselleştirme

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Biyoloji

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Fagositoz Erken Aşamaları görselleştirme

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

<meta charset="utf8"></head><body>Canlı Floresan, Hücre Karışıklığının Ters Görüntülenmesi ve Makropinositoz

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

Canlı Floresan, Hücre Karışıklığının Ters Görüntülenmesi ve Makropinositoz

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

"Fagozomun Kapatma Deneyi" kullanma Üç Boyutta fagozomun Formasyonu Visualize'a Toplam İç Yansıma Floresan Mikroskobu (TIRFM)

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

Canlı Makrofagların içinde Phagolysosome Biogenesis'den Çalışması

Makrofaj Fagositoz ve Aktin Polimerizasyon Değerlendirme Yüksek Verimli Florimetrik Tekniği

Oranlı metrik Floresan Mikroskopi fagosom pH ölçümü

Nötrofil fagozomun Görüntüleme ve Sitoplazma bir oranlı metrik pH Göstergesi kullanma

Fagositoz fare makrofajlar tarafından hızlandırılmış 3D görüntüleme

TIRF Mikroskopi ile Tek olay ebatları da G protein-bağlanmış alıcılar üzerinden Klatrin dolayımlı endositoz görselleştirme

Fagositoz Erken Aşamaları görselleştirme

Canlı Floresan, Hücre Karışıklığının Ters Görüntülenmesi ve Makropinositoz

Canlı Floresan, Hücre Karışıklığının Ters Görüntülenmesi ve Makropinositoz