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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, &#8805;98%&#160;</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 4mm</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>100mm 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&#160;</td>
			<td>TILL Photonics</td>
			<td></td>
			<td>&#160;Oil-immersion objective&#160;(N 100x, NA1.49.),&#160; heating chamber with CO2, 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&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td>Life technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td>Life technologies</td>
			<td>11835-105</td>
			<td>Warm in 37&#176;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&#176;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

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

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

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

Nötrofil Ekstrasellüler Tuzaklar: Onları nasıl oluşturun ve görselleştirin için

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

İmmünoloji ve Enfeksiyon

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

HIV ve Live Konfokal Mikroskop Floresan Klonlar kullanılarak HIV görselleştirme Hücre-hücre Transferi

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2-foton Mikroskop kullanarak Enfekte Akciğerler Nötrofiller Fagositoz ve NET-oluşumu Doğrudan Gözlem

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Toplam Protein Ekstraksiyon ve 2-D Jel Elektroforez Yöntemleri<em&gt; Burkholderia</em&gt; Türler

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Görselleştirme ve Miktarının Belirlenmesi için floresan proteinleri kullanarak<em&gt; Chlamydia</em&gt; Vakuol Büyüme Dinamikleri Yaşayan Hücrelerde

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

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 ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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 ...

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

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Oranlı metrik Floresan Mikroskopi fagosom pH ölçümü

Nötrofil fagozomun Görüntüleme ve Sitoplazma bir oranlı metrik pH Göstergesi kullanma