The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

Etik Beyanı: Tüm hayvan manipülasyonları Cenevre Üniversitesi Hayvancılık Araştırma Komitesi kurallarına sıkı göre yapıldı. Fagositik Hedeflerinin 1. Hazırlık <ol><li> 10 ml steril fosfat tamponlu salin (PBS) kurutularak zimosan 20 mg ekleyin. 10 dakika boyunca kaynar su banyosuna Vorteks ve ısı. 5 dakika boyunca 2.000 xg&#39;de Serin ve santrifüj. </li><li> Bir su banyo sonikatörü içinde 10 dakika için supernatant, 1 ml PBS içinde tekrar süspansiyon ve sonikasyon çıkarın. Iki adet 1,5 ml&#39;lik tüplere 500 ul aktarın. 5 dakika boyunca 11.000 x g&#39;de santrifüjleyin. </li><li> 500 ul PBS ile yıkama tekrarlayın. Kaynatıldı zimosan donduruldu ve -20 ° C&#39;de saklanır, bölünmüş olabilir. </li><li> Taze CO 3 0,1 M Na-2 hazırlanan 500 ul, pH 9,3, yukarıdaki (aşama 1,2) iki kez zimosan yıkayın. </li><li> Son yıkamadan sonra 490 ul süspanse. 10 mg 10 ul ekle / FITC dimetil sulfoxid içinde eritildi mle (DMSO), girdap, folyo ile kapatın ve oda sıcaklığında 4 saat boyunca çalkalanarak kuluçkalayın. </li><li> Önce 0.5 mi DMSO + 300 ul PBS, daha sonra 0.5 ml DMSO + 450 ul PBS, o zaman sadece PBS sonra + 150 ul PBS, 0.5 ml DMSO, yukarıda tanımlandığı gibi (aşama 1,2) yıkama, bağlanmamış boya kaldırın. </li><li> Bir hemositometre kullanılarak zymosan güvenin. 500 ul PBS, girdabına zimosan 1 ul ekleyin, o zaman merkezi ızgara üzerine yerleştirilen lamel karşılayan hemositometre odasına kenarına 10 ul ekleyin. 20X amacı ile donatılmış bir aydınlık alan mikroskop hemositometre monte edin. </li><li> 16 kareler içeren sol üst köşesindeki odaklanın ve bir el taksitli sayacı kullanarak bu alandaki tüm zimosan parçacıkları saymak. Sağ alt köşede tekrarlayın. = Zymosan konsantrasyon / 2 x 500 x 10 4 zymosan / ml Sayısı: aşağıdaki denklem kullanılarak zymosan konsantrasyonunu hesaplayın. Etiketli zimosan donduruldu ve -20 ° C&#39;de saklanır, bölünmüş olabilir. </li><li> Etiketli zymosan wi opsonize100 seyreltme: 1 bir tavşan anti-zimosan antikor th. Vorteks ve 37 ° C su banyosu içinde 1 saat boyunca inkübe edilir. (Adım 1.2) yukarıdaki gibi 500 ul PBS ile yıkayın. Not: zymosan sonra analiz daha zorlaştırabilir öbekler şeklinde eğilimi Sonikasyon yüksek, özellikle de opsonızasyonu sonra, tavsiye edilir. % 65 h / h sıçan ya da insan serumu ile Opsonizasyon alternatif olarak kullanılabilir. </li><li> Adımda 1,7-1,8 de tarif edildiği gibi, bir hemositometre kullanılarak zimosan Sayısı ve 100 x 10 6 parçacık / mL&#39;ye seyreltilir. Örneğin tamamlayıcı veya opsonızasyonu gibi diğer fagositik uyarıcıları ile Opsonizasyon alternatif gerçekleştirilebilir. </li></ol> 2. Canlı Video Mikroskopi <ol><li> Başka bir yerde 38 detaylı bir şekilde tarif edildiği gibi primer fare nötrofil izole edin. <ol><li> Alternatif olarak, herhangi fagositik hücre türünü kullanın. Roswell Park Memorial Institute (RPMI),% 5 fetal dana serumu (hacim / hacim), 200 U penisilin / streptomyci ile desteklenmiş 1640 ortam içinde buz üzerinde tutun nötrofillerin, 10 x 10 6 hücre / ml&#39;lik bir konsantrasyonda kullanıma hazır olana kadar. Diğer hücre tipleri 1-4 gün önce, tohumlanabilir. </li></ol></li><li> , 35 mm cam alt çanağın merkezine nötrofillerin 2 x 10: 5 (20 ul) ilave edilir ortam 50 ul ekleyerek damla yayılması ve hücrelerin yapışmasına izin vermek için 5 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Hank dengeli tuz çözeltisi (HBSS) içinde 1 mi belirli bir MPO inhibitörü 4-aminobenzoik hidrazid (4-ABH, 10 uM) ihtiva eden 37 ° C&#39;ye ısıtıldı ekleyin. Sodyum azit (5 mM) gibi Spesifik olmayan MPO inhibitörleri de kullanılabilir, ancak yakın zamanda, pH 39 ek spesifik olmayan etkiler gösterir uygulamak için önerilmiştir. </li><li> Bir 37 ° C ısıtma sistemi ve 40X petrol amacı ile donatılmış bir geniş açılı floresan mikroskop çanak monte edin. Hücreler ve ekipmanlar denge sağlamak için 10 dakika için görüntüleme hücrelere inkübe edin. </li><li> Parlak-alan iletim görüntü elde etmek için mikroskop ayarlarını yapın [phase ya da 440 nm ya da 490 nm eksitasyon ve 535 nm emisyonda, her 30 saniyede eğer varsa diferansiyel girişim kontrast (DIC)]. </li><li> Çok fazla photobleaching ve fototoksisite ve deneysel sorular bağlı olarak soruluyor önlemek için mikroskop sistemine göre pozlama süresini ve satın alma sıklığını ayarlayın. Görüntü elde etme başlayın. </li><li> 2 dakika sonra, 10 x 10 6 (50 ul), FITC-zimosan çanak merkezine opsonize ekleyin. Kullanılan fagosit ve hedefin tipine bağlı olarak hücre oranlarını: Hedef ayarlayın. </li><li> 30 dakika boyunca görüntülemeyi devam edin. 30 dakika sonra time-lapse edinimi durdurmak ve bir zamanlayıcı başlar. Sahne taşıyın ve 5 dakika içinde, görüntü diğer fagozomların bakış farklı alanlarda 10 veya daha fazla anlık yakalamak. </li></ol> 3. Kalibrasyon ve Kontrol Deneyleri <ol><li> Deney günü (tarifleri Tablo 1&#39;de tarif edilmektedir), önceki pH ayarlama solüsyonlarının hazırlanması ve 10 ml&#39;lik miktarlar halinde dondurma.Bir su banyosu içinde 37 ° C &#39;kalibrasyon çözümleri ve sıcak çözülme. Bir pH ölçer ile pH kontrol ve çözeltiler gerçek pH kaydedilir. </li><li> Önceki görüntü alımı için cam alt çanak peristaltik bir pompa monte edin ve (bölüm 2) Yukarıda açıklandığı gibi canlı video mikroskopi gerçekleştirin. </li><li> 30 dakika iktisap sürenin sonunda, yemeğin içindeki çözüm kaldırmak için pompa açın ilk kalibrasyon çözeltisinin 1 ml ekleyin ve pompayı durdurun. </li><li> Sinyal genellikle 5 dakika stabilize kadar bekleyin. Her kalibrasyon çözeltisi ile tekrarlayın. <ol><li> Deney, günde en azından bir kalibrasyon gerçekleştirin ve yüksek pH ile başlayan ve en sıralı olarak çalışma, hem de düşük pH başlayarak ve en yüksek doğru ardışık çalışma kalibre edin. </li></ol></li><li> Bir kontrol olarak, cam alt tabak içinde 900 ul HBSS içinde hücreler, HBSS içinde seyreltilmiş 100 uM NADPH oksidaz inhibitörü difenil propionium iyodür 100 ul (DPI) ekleyin ve10 dakika inkübe edilir. </li><li> Edinimi başlatın ve (adım 2.7) yukarıdaki gibi zymosan ekleyin. Fagositoz 15 dakika sonra HBSS içinde seyreltilmiş bir (conca) konkanamisin 10 uM V-ATPaz inhibitörü 10 ul ve 15 dakika daha devam görüntüleme. </li></ol> 4. Analiz <ol><li> Time-lapse film ve fagosom anlık analizi. Not: Aşağıdaki talimatlar ImageJ için özeldir. Adım adım talimatlar kullanılan yazılım bağlı olarak büyük ölçüde değişebilir, ancak bu bölümde anlatılan fonksiyonlar en profesyonel görüntü analiz yazılımı mevcuttur. <ol><li> Profesyonel görüntü analiz yazılımı ile görüntüleri açın. Hiçbir hücreler vardır bir alanda kare çokgen seçim aracıyla küçük bir kare yatırım getirisi çizim ve ROI Eklentiler&gt; BG Çıkarma tıklayarak 490 kanal arka plan çıkarın. Ardından&gt; tıklayarak Düzen&gt; Seçim 440 kanalda aynı yatırım getirisini yüklemek Seçme ve tekrarı geri yükleyin. </li><li>Daha sonra Operasyonu açılır menüden &#39;Divide&#39; seçerek, ... Süreç&gt; Görüntü Hesaplama tıklayarak Image1 ve image2 açılır menülerden uygun görüntüleri seçerek 440 kanala bölünür 490 kanal 32-bit oranı görüntü olun ve 32-bit (float) sonuç check-kutusunu işaretleyerek. </li><li> &gt; Rainbow RGB Görüntü&gt; Arama Tabloları tıklayarak bir oran ile uyumlu renk kodlama renk kodlama look-up tablosu değiştirin. Eşik oranı görüntü&gt; Threshold&gt; Görüntü tıklayarak 0 ve sonsuzluk piksel ortadan Ayarlanır .... NaN onay kutusunu Set arka plan piksel seçili olduğundan emin hale o zymosan kırmızı görünür, böylece kaydırma çubuklarını ayarlayın ve Uygula&#39;yı tıklatın. </li><li> Görüntü tıklayarak bir çok kanallı kompozit içine parlak alan kanalı ile bu oran yığını birleştirin&gt; Renk&gt; Kanallar Birleştirme ve gri kanal açılır menüden parlak alan görüntü ve yeşil kanal açılır menüden oran görüntüsünü seçme ve seçmeOnay kutusunu kaynak görüntüleri tutun. Time-lapse film veya fagozomların analiz edilmelidir belirlemek için anlık tarayın. Parlak alan kanal için yararlıdır. </li><li> Ekle&gt; fagozomların etrafında oval poligon seçim aracını kullanarak ilgi (ROI) bölgelerini çiziniz ve Analiz&gt; Araçlar&gt; ROI Yöneticisi tıklayarak ROI yöneticisi ekleyebilirsiniz. Daha fazla&gt; Çok ölçün ve dilim onay kutusunun başına bir satır de-seçilerek tıklayarak kendi zymosan yoğunluk oranını ölçün. </li><li> Time-lapse film için, parça her zaman noktasını ölçmek için Ctrl + M yazarak, bir yatırım getirisi çizim ve zamanla yoğunluk değerlerini izlemek için gerektiğinde yatırım getirisi hareket ettirerek bir seferde bir fagozomların. Bir elektronik tablo yazılımı içine Sonuçları penceresinde ölçümleri kopyalayın. Not: bağımsız deneyler 15 (deney x 3 başına 5 zamanlı kurslar) Analizi ideal ama daha az ya da gözlenen değişkenlik bağlı gerekebilir olduğunu. İB&#39;leri Tasarruf her zaman tavsiye edilir. Bazı yazılım paketleri izin görüntü regiStration ve İB&#39;nin dinamik güncelleştirme, analiz bu bölümünü kolaylaştırmak. </li></ol></li><li> Floresans pH değerlerine Dönüşüm <ol><li> Bölüm 4.1&#39;de açıklandığı gibi kalibrasyon time-lapse filmlerde fagozomların için 490/440 oranını ölçün. </li><li> Elektronik tabloda zamanla oran değerlerini çizmek ve her pH kalibrasyonu karşılık gelen süre ortasında içinde 5 zaman noktalardan (5-20 arası, genellikle) görüş alanı içindeki tüm fagozomların ortalama oran değerlerini hesaplamak Çözelti. (Ayrıca Şekil 5A kırmızı kutuları bakın.) </li><li> Gerçek ölçülen pH karşı bu ortalama 490/440 oranı değerlerini çizilir. Tek bir grafiğe içine en az 3 bağımsız kalibrasyon birleştirin. Genellikle sigmoid denkleme, doğrusal olmayan regresyon (en iyi uyum eğrisi) gerçekleştirmek için bir istatistik veya sayısal bir yazılım paketi kullanın. Not: Örneğin, Şekil 5B&#39;de kullanılan denklem Boltzmann sigmoidal oldu [Y = Taban + ((Top-Taban)/ (1 + EXP ((V50-X) / Eğim)) Y değerleri 490/440 oranları], X pH değerleri ve Üst, Alt, V50 ve eğim parametreleri yazılım tarafından hesaplanır vardır. </li><li> PH oranı değerleri dönüştürmek için elde edilen denklemi kullanın. Not: Örneğin, Şekil 5B&#39;de, sodyum azid mevcudiyetinde kalibrasyon eğrisi için elde edilen denklem şöyledir 490/440 oranı = 1,103 + [(2,89-1,103) / (1 ​​+ EXP ((6,993 pH) /0.9291)] . pH Çözme aşağıdaki denklemi sağlar: pH = 6,993-0,9291 * [LN ((1,178 / (Oran-1,103)) - 1))]. </li></ol></li></ol>

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+activates+virulence+gene+transcription+within+acidified+macrophage+phagosomes.">Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.</a> Proc Natl Acad Sci U S A. 89 (21), 10079-10083 (1992).</li><li>Lukacs, G. L., Rotstein, O. 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=Determinants+of+the+phagosomal+pH+in+macrophages.+In+situ+assessment+of+vacuolar+H(+)-ATPase+activity,+counterion+conductance,+and+H++'leak'.">Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ 'leak'.</a> J Biol Chem. 266 (36), 24540-24548 (1991).</li><li>Watanabe, K., Kagaya, K., Yamada, T., Fukazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+candidacidal+activity+in+macrophages+activated+by+recombinant+gamma+interferon.">Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.</a> Infect Immun. 59 (2), 521-528 (1991).</li><li>Ip, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+phagosome+acidification+are+required+for+pathogen+processing+and+MyD88-dependent+responses+to+Staphylococcus+aureus.">Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus.</a> J Immunol. 184 (12), 7071-7081 (2010).</li><li>Clarke, M., Maddera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocyte+meets+prey:+uptake,+internalization,+and+killing+of+bacteria+by+Dictyostelium+amoebae.">Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae.</a> Eur J Cell Biol. 85 (9-10), 1001-1010 (2006).</li><li>Jankowski, A., Scott, C. C., 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=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomal+proteolysis+in+dendritic+cells+is+modulated+by+NADPH+oxidase+in+a+pH-independent+manner.">Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.</a> EMBO J. 31 (4), 932-944 (2012).</li><li>Mantegazza, A. R., 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=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112 (12), 4712-4722 (2008).</li><li>Balce, D. R., Allan, E. R., McKenna, N., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., 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+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

Daha fazla zaman hedefleri bağlanmış bir pH duyarlı boya kullanılarak benzer bir strateji geliştirerek, ancak fagozomların bir popülasyonunun ortalama pH ölçer gibi spektroskopi ve FACS gibi alternatif yöntemler, daha zaman alıcı olmakla birlikte, mikroskopi çeşitli avantajlar sunmaktadır. İlk olarak, iç ve dış içsel bağlı değil, diğer bir deyişle, partiküllerin kolayca sırasıyla gidermek ya da dış parçacıklar etiket, örneğin tripan mavisi ya da antikorlar gibi diğer kimyasallar eklem...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table><tbody><tr><td>Zymosan A powder</td><td>Sigma-Aldrich</td><td>Z4250</td><td>Various providers exist</td></tr><tr><td>Fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>F7250</td><td>Various providers exist</td></tr><tr><td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td><td>Life Technologies</td><td>Z2850</td><td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td></tr><tr><td>4-Aminobenzoic hydrazide (4-ABH)</td><td>Santa Cruz</td><td>sc-204107</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Diphenyleneiodonium chloride (DPI)</td><td>Sigma-Aldrich</td><td>D2926</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Concanamycin A (ConcA)</td><td>Sigma-Aldrich</td><td>27689</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Nigericin</td><td>Sigma-Aldrich</td><td>N7143</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Monensin</td><td>Enzo</td><td>ALX-380-026-G001</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Life Technologies</td><td>14200-075</td><td>Toxic, use gloves, various providers exist</td></tr><tr><td>Hank&#039;s balance salt solution</td><td>Life Technologies</td><td>14025092</td><td>Ringer&#039;s balanced salt solution or other clear physiological buffers may be substituted.</td></tr><tr><td>Sodium carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S7795</td><td>Various providers exist</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M3671&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td>Various providers exist</td></tr><tr><td>N-Methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>3777</td><td>Various providers exist</td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Sigma-Aldrich</td><td>T1503&amp;nbsp;</td><td>Various providers exist</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9333</td><td>Various providers exist</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td>Various providers exist</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M8266</td><td>Various providers exist</td></tr><tr><td>Absolute Ethanol (EtOH)</td><td>Sigma-Aldrich</td><td>2860</td><td>Various providers exist</td></tr><tr><td>Glass-bottom 35 mm petri dishes (Fluorodish)</td><td>World Precision Instruments</td><td>FD35-100</td><td>Ibidi &amp;micro;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td></tr><tr><td>Sonicating water bath</td><td>O. Kleiner AG</td><td></td><td>A sonicator may be used instead, various instrument providers exist</td></tr><tr><td>Heamocytometer</td><td>Marienfeld GmbH</td><td></td><td>Various instrument providers exist</td></tr><tr><td>Widefield live imaging microscope</td><td>Carl Zeiss AG</td><td></td><td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Peristaltic pump (Dynamax RP-1)</td><td>Rainin</td><td></td><td>Various instrument providers exist</td></tr><tr><td>pH meter</td><td>Schott Gerate GmbH</td><td></td><td>Various instrument providers exist</td></tr><tr><td>Manual Counter</td><td>Milian SA</td><td></td><td>Various instrument providers exist</td></tr></tbody></table>

measuring phagosome ph by ratiometric fluorescence microscopy

Fagositoz doğuştan gelen bağışıklık hücreleri öldürmek veya etkisiz yutulur malzeme veya antijen olarak sunmak ve adaptif immün yanıtları başlatmak için bakteri, apoptotik hücreler veya diğer yabancı partikülleri yutmak hangi aracılığıyla temel süreçtir. Fagozomların pH endomembranes ve proteolitik enzimler, fagositik vakuol bir parçalayıcı organele içine olgun izin olaylar aktivasyonu ile fizyon ya da füzyon düzenleyen kritik bir parametredir. Buna ek olarak, H + translokasyon diğer konakçı dokulara etkili bir öldürme için önemli ve sinyal olan reaktif oksijen türlerinin (ROS), yüksek seviyelerinin üretimi için gereklidir. Birçok hücre içi patojenler, phagosomal asitlenmesine sınırlayıcı fagosom biyoloji pH önemini vurgulayarak fagositik öldürmeyi yıkmak. Burada floresein izotiosiyanat (FITC) kullanılarak nötrofillerde phagosomal pH ölçmek için bir yöntem tarif rasyometrik fagositik targ olarak zimosan -etiketliets ve canlı hücre görüntüleme. Deney 490 nm&#39;de uyarıldığında, ancak tek bir boyanın, yerine mutlak floresandan daha, bir pH bağımlı oranının sayılmasına olanak tanıyan, 440 nm&#39;de uyarıldı olmadığında asidik pH ile söndürülür FITC, floresan özelliklerine dayanır. In situ boya kalibrasyonu ve gerçek pH değerlerine oranı dönüştürme gerçekleştirmek için ayrıntılı bir protokol, aynı zamanda temin edilmektedir. Onlar böyle ağartma olarak tedirginlikler daha az duyarlı olarak tek boya rasyometrik yöntemleri genellikle tek dalga boyu veya çift boya sözde rasyometrik protokollere üstün kabul edilir, ölçülen sinyali tahrif değişiklikleri, lazer varyasyonları ve düzensiz etiketleme, odak. Bu yöntem, kolaylıkla diğer fagositik hücre tiplerinde pH ölçmek için modifiye edilebilir ve zymosan atıl tanelerden canlı mikroorganizmalara, diğer herhangi bir amin içeren bir parçacık ile ikame edilmiş olabilir. Son olarak, bu yöntem, farklı pH aralıklarında ya da diğer phagosom karşı duyarlı olan diğer floresan probları faydalanmak için adapte edilebiliral faaliyetleri, bu fagozomların fonksiyonel görüntüleme için genel bir protokol yaparak.

Aşağıda bir deney için temsili sonuçlar olduğu takdirde burada yabani tip ya da kemik-iliğinden izole edilmiş primer fare nötrofil phagosomal pH Hvcn1 - / - farenin karşılaştırıldı. Başarılı bir deneme için, daha sonra görüntü analizi sırasında segmente daha zor olacaktır çok fagozomların, kaçınarak, time-lapse film tüm süresince görüş alanı içinde yeterli fagozomların elde etmek önemlidir. Şekil 1&#39;de görüldüğü gibi İyi ve k...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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 ...

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Immunology and Infection

13.9K Görüntüleme.  University of Geneva. 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. 

Video: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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 ...

İmmünoloji ve Enfeksiyon

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 ...

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Efferocytosis tek hücreli floresans mikroskobu tarafından miktar

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

Çift Florofor Oranımetrik Mikroskopi kullanılarak Makropinozomların pH, Redoks Kimyaları ve Bozunabilir Kapasitesinin Ölçülmesi

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Konfokal mikroskopi kullanılarak Yaşayan hücreler içinde endositik ve Sitosolik Bölümleri eşzamanlı pH Ölçümü

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Biyoloji

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

İPSC-Makrofajlar tarafından Ölü Nöroblastom Hücrelerinin Fagositozu için İn vitro Nicel Görüntüleme Tahlili

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Nörobilim

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin etiketli Reseptör kullanarak Subselüler Yerelleşme ve Ticareti Monitör

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

For non-covalent fixation of the SRBCs, pour 2 milliliters of the IgG-opsonized cells into each of the poly-lysine-coated dishes, and centrifuge the samples in a swinging rotor centrifuge. Discard the supernatants, and wash the particles one time with 2 milliliters of PBS plus 10% BSA.
Then, incubate the particles with 2 milliliters of fresh PBS plus 10% BSA for 30 minutes at room temperature, followed by three washes with 2 milliliters of PBS. After the last wash, replace the PBS with 2 milliliters of 37 degrees Celsius serum-free microscopy medium.
Place an SRBC-coded dish onto the microscope stage. Then, scrape the transfected cells of interest from the bottom of the culture dish and pipette the cells a few times to achieve a single-cell suspension. Add the transfected cells to the SRBC-coated dish.
Start the live acquisition software. Find a cell that expresses the fluorescently tagged proteins and adjust the position of the dish so that a cell of interest is in the middle of the field. Acquire 500 images at different angles from 0 to 5 degrees at 0.01-degree increments at one excitation wavelength.
To determine the critical angle for the incident light to be totally reflected at the glass-medium interface and generate an evanescent wave, open the image sequence in the appropriate imaging software. Select a region of interest in the cell with uniform fluorescence.
Then, under the Image tab, select Stacks and plot Z-axis profile. To plot the Z-axis profile mean fluorescence intensity measured in the region of interest with the function of the angles on the X-axis. Any angle value on the x-axis superior to the critical angle can then be used during the microscopy session to obtain a TIRF signal.

Oranlı metrik Floresan Mikroskopi fagosom pH ölçümü

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