Name: phagosome migration and velocity measured in live primary human macrophages infected with hiv-1
Uploaded: 2016-09-05T15:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

protokol, ulusal ve uluslararası mevzuata ve yerel yönetmeliklere tam uyum içinde yapılmalıdır. araştırma amaçlı kan bağışı için rızalarını verdi sağlıklı donörlerden kan Kurumları anlaşma imzaladı hangi ile Kan Transfüzyon Merkezleri elde edilmiştir. İnsan kanı kullanılması özel bir koruma alınmalıdır. HIV-1 ile yapılan deneyler 3 veya 2 (BSL-3 veya 2) laboratuvar, yerel mevzuata uygun bir biyogüvenlik seviyesinde yapılmalıdır. Yapışma yoğunluk gradyan santrifüjü ve seçim İnsan monosit türevli makrofajlar (hMDMs) hazırlanması 1. <ol><li> Sağlıklı vericilerden (9 mi) taze kan başla. 70 ml&#39;lik bir son hacim elde etmek için Ca + 2 ve Mg + 2 olmaksızın, steril 1 x fosfat tamponlu tuzlu su (PBS) ile taze kan tüm hacmi seyreltilir ve hafifçe iki 50 ml konik tüp içine seyreltilmiş kan eklemek (tüp başına 35 mi) bir 15 ml üstündekieutral, yüksek her bir tüp içinde daha önce çözelti içinde, yüksek kütle, hidrofilik polisakarit dallanmış. </li><li> fren olmaksızın 20 ° C de 20 dakika boyunca 537 x g&#39;de santrifüje kan her iki tüp. Daha sonra ara yüzeyde parçalı hücre halkasında bulunan periferal kan mononükleer hücreleri (PBMC&#39;ler) toplamak ve Ca + 2 ve Mg + 2 olmadan 1x 15 ml PBS içeren yeni bir 50 mL tüp içine transfer edin. </li><li> Santrifüj 20 ° C&#39;de 5 dakika boyunca 218 x g&#39;de hücreleri Ca + 2 ve Mg olmayan 1x PBS 45 ml pelet 2+. </li><li> Santrifüj 20 ° C&#39;de 5 dakika boyunca 218 xg&#39;de hücreleri, Ca + 2 ve Mg + 2 olmadan 1x PBS, 10 ml pelet tekrar süspansiyon ve Tripan Mavisi 1/200 nihai seyreltme seyreltilerek hücreleri sayın. </li><li> 20 ° C&#39;de 5 dakika boyunca 218 x g&#39;de hücreleri santrifüj ve 1640 ortamında 2 mM L-glutamin ve 100 ug / ml Penic ile desteklenmiş RPMI (Roswell Park Memorial Institute) peletillin-streptomisin, 6 gözlü bir plakanın her bir gözünde ortam 2 ml oyuk başına 7 x 10 6 PBMC&#39;ler için. </li><li> 2 saat boyunca% 5 CO2 ile 37 ° C &#39;de inkübe edin. 2 saat sonra monositler plastik yapışmış olur. </li><li> Taze izole edilmiş monositlerin, hMDMs içine ayırt orta aspire ile takviye edilmiş 2 ml hMDM ortamı (RPMI 1640, fetal buzağı serumu (FCS decomplemented% 10), 2 mM L-glutamin ve% 1 penisilin-streptomisin) ile değiştirmek için izin vermek için rekombinant insan makrofaj koloni uyarıcı faktör, 10 ng / ml&#39;lik bir son konsantrasyonda (RHM-CSF) vardır. </li><li> 11 gün sonra (Şekil 1),% 5 CO2 ile 37 ° C &#39;de inkübe hücreleri. </li><li> 11. günde de orta kaldırmak ve oyuk başına soğuk hMDM ortam, 2 ml ile 2 kez yıkanır. Sonraki soğuk 1 x PBS, 1 ml her bir çukura 2 kez yıkayın. </li><li> (2 mM ethylenediaminetetraacetatic asit ile, soğuk 1 x PBS, 1 ml her bir 1 kez yıkama, tam olarak farklılaşmış hMDMs ayırmak içinEDTA) ve 4 ° C&#39;de 15-60 dakika boyunca oyuk başına 2 mM EDTA, soğuk 1 x PBS, 2 ml hücreleri inkübe edin. NOT: Hücreler var ayırmak için alternatif yöntemleri (örneğin, tripsin veya tripsin benzeri faaliyetler) iyi sonuçlar 11 verebilir. </li><li> (Yukarı ve aşağı kuyuda hafifçe pipetleme tamamlandı) ayrıldıktan sonra, hücreleri toplamak ve soğuk hMDM orta 10 ml içeren buz üzerinde bir 50 ml tüp koyun. </li><li> Santrifüj 20 ° C&#39;de 5 dakika boyunca 218 x g&#39;de hücreler soğuk hMDM ortamı 10 ml pelet tekrar süspansiyon ve Tripan Mavisi 1/20 seyreltilerek hücreleri sayın. </li><li> 35 mm mikroskopi dereceli cam alt tabak başına 1 x 10 6 hMDMs hücreleri tohum ve 1 gün için% 5 CO2 37 ° C&#39;de inkübe edin. </li></ol> HIV-1 viral Hisse Senedi 2. Üretim ve Niceleme Not: NLR4.3 HIV-1 Gag-iGFP (yeşil floresan protein) M. SCHINDL bir R5-tropik zarf, hediye taşıyanER 10 makrofajlan enfekte etmek ve gerçek zamanlı olarak enfekte olan hücreleri görmek için kullanılmaktadır. <ol><li> Ticari bir transfeksiyon reaktifi kullanılarak karşılık gelen proviral DNA 6 ug insan embriyonik böbrek 293T hücreleri (2 x 10 6, 100 mm çanak) transfeksiyonu ile viral stoklar üretir. </li><li> hücreler ve sayılmasına yönelik bir beta-galaktosidaz renk ardından viral stoklar seri dilüsyonları kullanılarak (HIV-1 LTR kontrolü altında beta-galaktosidaz genini taşıyan) HeLa tzm-BL göstergesi hücreleri kullanılarak virüs stoklarının enfeksiyon niceliğini mavi hücreler 12. </li></ol> HIV-1 ile hMDMs 3. Enfeksiyon <ol><li> hMDMs (1.13 kaplanmıştır hücreler) enfeksiyon (MOI) bir çokluğunda hMDM ortamının 0.02-0.03 1 ml virüs ekleyin. Kuyular hMDM ortamının sadece 1 ml ilave kontrol ve 2 gün (Şekil 1),% 5 CO2 ile 37 ° C &#39;de yemekler kuluçkalanması. </li><li> günde 2 hMDM Med hücreleri yıkayın3 kez amonyum ve tabak başına taze hMDM ortamının 1 ml. 6 gün (Şekil 1),% 5 CO2 ile 37 ° C &#39;de inkübe hücreleri. </li></ol> Koyu kırmızı kan hücrelerine 4. Opsonizasyon <ol><li> Tabak başına 7 x 10 6 SRBClere hazırlanması için, 4 dakika boyunca 600 x g&#39;de santrifüj ile 1x PBS içinde% 0.1 Sığır Serum Albumin (BSA) ihtiva eden çözeltinin 100 ul SRBClere iki kez yıkayın. </li><li> SRBClere 5 ul başına alt aglütinasyon konsantrasyonda tavşan IgG, anti-SRBClere PBS / BSA% 0.1 1 x 500 ul yıkandı SRBClere yeniden süspanse edin ve 30 dakika boyunca oda sıcaklığında dönme ile inkübe edilir. Not: anti-SRBC&#39;ler IgG alt aglütinasyon konsantrasyonunu belirlemek 96 oyuklu bir plaka içerisinde 20 ul 1 / 25.600 1/50 arasında (13.1 mg / ml stok konsantrasyonunda) IgG seri dilüsyonları hazırlanması. Her oyuktaki 20 ul 2 x 10 6 SRBClere ekleyin ve birkaç saat boyunca, karanlık bir odada tabak koydu. alt aglütinasyon konsantrasyonudurhemen önce iyice aglütinasyon (IgG + SRBC&#39;ler bir ağ oluşturan) ile kuyu seyreltme. </li><li> Dönme sonra 4 dakika boyunca 600 x g&#39;de IgG opsonize-SRBClere santrifüj ve 4 dakika için 600 x g&#39;de santrifüj ile 1X PBS / BSA% 0.1, 100 ul ile yıkayın. </li><li> 2 mM L-glutamin ve% 1 penisilin-streptomisin (1 ml / kutu) ile desteklenmiş önceden ısıtılmış fenol kırmızısız RPMI ortamında IgG opsonize-SRBClere yeniden süspanse edin. </li></ol> 5. Canlı Hücre video Mikroskopi Fagositoz Assay <ol><li> Bu CO2 nemlendirme su ile bir şişe içinden geçen 37 ° C&#39;de bir ısıtma odası ile donatılmış bir döner disk sistemi gibi bir konfokal görüntüleme sistemi kullanın. </li><li> fagositoz testinde başlangıcından önce 37 ° C &#39;de mikroskop aşamasında için deney öncesinde ısıtma odasına açın. mikroskop ve bilgisayarı açın ve görüntüleme yazılımı yükleyin. </li><li> Böyle tarama hızı, Magnificat gibi görüntüleme ayarlarını optimizeiyon, çözünürlük, vs 60 ila 120 dakika arasında her dakika sahada başına ve görüntü tek bir karede en az bir hücre var. NOT: Burada, örnek 63X lens ile 60 dakika boyunca bir kare her dakika görüntülü. </li><li> Mikroskop sahnede görüntüleme çanak yerleştirin. Odağı ayarlamak ve konum alanına sadece bir bütün HIV-1 ile enfekte makrofaj bulmak için. kullanılan görüntüleme sistemi ve prob göre uygun uyarma / emisyon ayarlarını kullanın. Fagozomların gözlemlemek için parlak bir alan (BF) kanalı ekleyin (Şekil 1ii). iletilen ışık ve maruz kalma süresi yüzdesini ayarlayarak farklı kanal görüntülerin görünümünü optimize edin. NOT: Burada, NLR4.3 HIV-1 Gag-iGFP lazer% 20 (Şekil 1i) ile zaman maruz kalma 50 ms ile 491 nm lazer kullanarak heyecanlıydı. </li><li> Görüntüleme tabağı çıkarın ve çanağın 7 x 10 6 SRBC&#39;ler / ml (Şekil 1) SRBC süspansiyonu 1 ml. </li><li> 500 xg&#39;de santrifüjOda sıcaklığında 2 dakika, fagositozu senkronize bu santrifüj sonunda zamanını kaydetmek ve sahneye çanak dönün. </li><li> en az 1 saat süreyle her dakika - (genellikle 20 uçakları 0.3 mikron adım mesafe ile hücre kalınlığı boyunca) Z-yığınlarının içinde odaklama ve çekim GFP ve BF görüntüleri optimize. kullanılan görüntüleme sisteminin yerel dosya formatında zaman atlamalı video kaydetmek. NOT: Burada, zaman atlamalı filmleri yerli formatında kaydedilen, * .stk dosyaları. </li></ol> Time-lapse Filmler 6. Analizi <ol><li> video düzenleme için, menü &quot;Uygulamalar&quot; aşağı ve video düzenleme yazılımı sekmesi &quot;Değerlendirme Çok Boyutlu Veriler&quot; konulu damla tıklayın. <ol><li> Dosyayı açmak için, ardından &quot;Dizin Seç&quot; &quot;Select Bankası Dosyası&quot; üzerine tıklayın. &quot;Veri setleri&quot; kutusuna, (Kara Delikler formatında) analiz ve &quot;Görünüm&quot; tıklayın edinimi seçin. veri sütunu An zamanla bir tablo temsil edilmektedirdoğrultusunda d Z-planı. </li><li> Z-projeksiyon (Şekil 2, sol panel) enfeksiyonu temsil etmek, &quot;Dalgaboyları&quot; kutusuna 491 nm dalga boyu seçmek ve &quot;tüm uçakları&quot; ile &quot;Z projeksiyon&quot; sekmesine tıklayın. </li><li> (Bir zaman dizisini analiz &quot;Dalgaboyları&quot; kutusuna BF dalga boyunu seçin ve dış SRBClere (Şekil 2, kırmızı ok ucu), iç SRBC&#39;ler (Şekil 2, kırmızı daire) ve çekirdeği ayırt etmek için Z-ekseni üzerinde optimum düzlemi seçmek için Şekil 2, mavi daire). </li><li> video montajı kaydetmek için, &quot;Seçim [X]&quot; sekmesini ve ardından &quot;Load Image (lar)&quot; üzerine tıklayın. Son olarak, .tif formatta yüklü görüntüleri kaydetmek ve ImageJ yazılım sonraki açın. </li></ol></li><li> Çekirdeğin konumunu ve BF kanalda gözlenen farklı fagozomların (Şekil 3) ölçmek için ImageJ yazılım üzerinde eklenti &quot;Manuel İzleme&quot; kullanın. <ol><li> DowImageJ sitesinde &quot;Manuel İzleme&quot; eklentisi nload. ImageJ üzerinde (Şekil 3, aşama 1 ve 2) eklentisi ve görüntü dizisi analiz edilecek açın. </li><li> Böyle komşu kareler arasındaki süreyi temsil eden &quot;Zaman Aralığı&quot;, ve piksel başına mesafeyi temsil eden &quot;x / y kalibrasyon&quot; (Şekil 3, adım 3) gibi ayarları girin. NOT: 63X zoom ve 6.45 x 6.45 mikron 2 piksel boyutunda bir kamera kullanıldığı için Burada, her kare ve 0.205 mikron x / y kalibrasyon arasında 1 dakikalık bir zaman aralığı ile görüntü dizisi kaydedin. </li><li> Izleme başlatmak için, (Şekil 3, adım 4) &quot;Add track&quot; üzerine tıklayın ve içselleştirildiği zaman ilk kez bir SRBC merkezinde tıklayın. Bir sonraki kare otomatik olarak görüntülenir. </li><li> Süre içinde farklı pozisyonlarda (Şekil 3, kırmızı kutu içinde adım 6) sahip tüm karelerde SRBC merkeze tıklayın devam edin. <ol><li> kendi merkezinde tıklayarak bütün karelerde konumunu var çekirdeği izlemek için başlayın. Sonraki zaman SRBClere fonksiyonu farklı onların içselleştirme (kare), en tek tek (kendi merkezine tıklayarak) fagozomların izleyebilirsiniz. Kolaylık BF kanalda SRBC görmek için, izleme (Şekil 3, adım 5) sırasında &quot;Parlaklık ve kontrast&quot; penceresini kullanın. </li></ol></li><li> Her SRBC izleme arasında, yeni bir parça var (Şekil 3, adım 4) &quot;Add track&quot; üzerine tıklayın. Izleme sayısı ikinci sütunda değişmiş olacak, sonuç tablonun &quot;Parça n °&quot; (Şekil 3, adım 6). </li><li> Analizin bir sonraki adıma devam etmek için bir e-tablodaki verileri kaydetmek. </li></ol></li><li> (Çekirdek ve SRBClere içselleştirilmesi sonra ilk 5 dakika boyunca fagozomların hızı doğru SRBClere içeren fagozomların bir seyahat mesafe hesaplamak için Fi tablo yazılımını kullanınşekil 4). <ol><li> tablo tablo ve yeni bir tablo dosyasını açın. Bu yeni Aşağıdaki parametreler sadece dosyaya, zaman, x-içine aktarın ve çekirdek ve tüm SRBClere (Şekil 4A) koordinatı y. </li><li> Sadece kendi koordinatlar ile SRBClere ve çekirdek arasındaki mesafeyi hesaplamak XY koordinat düzleminde (Şekil 4B) çekirdeği ve SRBC dikkate alın. Not: iki nokta arasındaki mesafe onları bağlayan yolun uzunluğudur. düzlemde, SRBC ve çekirdek arasındaki mesafe Pisagor teoremi ile verilir. <ol><li> C (nukleus ve SRBC arasındaki mesafe) hipotenüs ve a uzunluğunu gösterir b diğer iki tarafın uzunluklarını göstermek durumunda, Pisagor denklemi olarak Pisagor teoremini ifade: <img alt="denklem 1" src="/files/ftp_upload/54568/54568eq1.jpg" /> Not: Aynı zamanda, Ortonormal üzerinde yatay mesafe(x çekirdeği -x SRBC) ve dikey mesafe b (SRBC -y y çekirdeği) &#39;dir. </li><li> Böylece, çekirdek ve SRBC (Şekil 4A, mor kutu) ile arasındaki mesafeyi hesaplamak: <img alt="denklem 2" src="/files/ftp_upload/54568/54568eq2.jpg" /> NOT: mikron mesafe olması x / y kalibrasyon (piksel) elde mesafe değeri çarpın. Burada, x / y kalibrasyon 0.205 mm. </li><li> Her zaman, çekirdek ve başlangıç ​​mesafesi (Şekil 4C) için SRBC arasındaki mesafeyi çıkarın. </li><li> İlk 5 dakika için zamana karşı ölçüm çizilir. Arsa doğrusal eğilim çizgisi (Şekil 5A) uygulayın ve SRBC içselleştirme sonra ilk 5 dakika boyunca çekirdeğin doğru fagosom hızını temsil doğrusal trend çizgisinin eğimini belirler. </li><li> ortalama ve istatistiki hatayı hesaplamak için veri harmanlamakhız değerleri uygun yazılımı (Şekil 5B) kullanılarak uygun bir biçimde bunları çizmek ve. </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

Bu teknik pek çok kritik adımlar vardır. enfeksiyon yüzdesi donör bağımlı olduğundan İlk olarak, HİV-1 ile hMDMs hazırlanması ve enfeksiyon önemlidir. Potansiyel in vivo virüs tarafından karşılaşılan makrofajlar durumu iyi şimdiye kadar karakterize edilmemiştir, çünkü notun, biz, enfeksiyon öncesinde in vitro polarize olmayan makrofajlar kullanmaya karar verdik. Bu makrofajlar de M1 veya M2 olduğunu gösteren çok sayıda yüzey markerlerinin ifadesiyle kontrol ve CD4 ve CCR5...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

<table><tbody><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td>For viral production</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>For acquisition</td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Thermo Fischer Scientific</td><td>Corning. Inc. 353934</td><td>For human monocyte-derived macrophages</td></tr><tr><td>Ficoll-Plaque PLUS</td><td>Dominique Dutscher</td><td>17-1440-03</td><td>a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>RT</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) 1x, liquid (High Glucose)</td><td>GIBCO, Molecular probes</td><td>31966-021</td><td>Conserved at 4 &amp;deg;C ; for HEK cells culture</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td>Conserved at 4 &amp;deg;C; for hMDMs culture</td></tr><tr><td>F&amp;oelig;tal Calf Serum (FCS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF0001</td><td>Conserved at -20 &amp;deg;C ; decomplemented</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Thermo Fischer Scientific</td><td>15140-122</td><td>Conserved at -20 &amp;deg;C ; for hMDMs culture</td></tr><tr><td>RPMI 1640 medium, no phenol red (10x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use ; for phagocytosis assay</td></tr><tr><td>FuGENE6 Transfection Reagent</td><td>Promega</td><td>E2692</td><td>Conserved at 4 &amp;deg;C ; for viral production</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><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td>Conserved at 4 &amp;deg;C</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>Conserved at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope DMI600</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal Spinning Disk Unit&amp;nbsp; CSU-X1M1</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>491 nm 50 mW laser</td><td>COBOLT CALYPSO</td><td></td><td></td></tr><tr><td>HCX PL APO CS Objectif</td><td>Leica</td><td></td><td>Objective lens ; Magnification 100X ; Numerical aperture 1.40 ; Immersion oil</td></tr><tr><td>CoolSnap HQ2 (FireWire) Camera</td><td>Photometrics</td><td></td><td>Pixel size 6.45 &amp;micro;m x 6.45 &amp;micro;m ; Definition 1,392 x 1,040 ; Encoding the image in 14 Bit</td></tr><tr><td>Metamorph 7.7.5 software</td><td>Molecular Devices</td><td></td><td>For the control of the microscope</td></tr><tr><td>GraphPad Prism software</td><td></td><td></td><td>For the statistics analysis</td></tr></tbody></table>

phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

HIV-1 ile enfekte edilmiş ve enfekte olmayan hMDMs ile fagositozu FcR aracılı modeli hedefleri olarak IgG opsonize SRBClere (Şekil 1) kullanılarak burada tarif edilmektedir. Bu protokol kritik adım hMDMs hazırlanması ve HIV-1 enfeksiyonu bulunmaktadır. Gerçekten de, farklı makrofaj verim ve kalite donörler arasında değişir, ve% 10-40 aralığında verimliliği ile enfeksiyon oranı. Buna ek olarak, IgG-opsonize-SRBClere hazırlanması Bu, tanıma neden ve ...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1 ile infekte Canlı İlköğretim İnsan Makrofagların Ölçülen fagosom Göç ve Hız

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Research

JoVE Journal

Immunology and Infection

8.1K Görüntüleme.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

Video: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

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 ...

İmmünoloji ve Enfeksiyon

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 ...

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 ...

"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.

"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 ...

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 ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Konfokal Mikroskopi ile Leishmania Fagositozunun İncelenmesi

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG-opsonize Kırmızı Kan Hücrelerinin Makrofaj Fagositozunun Gerçek Zamanlı Görüntülenmesi

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

İmmünoloji

İmmünodiyagnostik

Özelleşmiş Analizleri

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://app.jove.com/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

HIV-1 ile infekte Canlı İlköğretim İnsan Makrofagların Ölçülen fagosom Göç ve Hız

Özet

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

Fagositoz fare makrofajlar tarafından hızlandırılmış 3D görüntüleme

Konfokal Mikroskopi ile Leishmania Fagositozunun İncelenmesi

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

IgG-opsonize Kırmızı Kan Hücrelerinin Makrofaj Fagositozunun Gerçek Zamanlı Görüntülenmesi

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure