This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

10 nm Altın nanopartiküller (Altın NPS) 15 1. sentezi <ol><li> Çözelti A hazırlayın: 160 mi Milli-Q, ya da iki kez damıtılmış su içine 2 ml% 1 sulu bir altın klorür ekleyin. </li><li> Çözelti B hazırlanması: 32 mL Milli-Q, ya da iki kez damıtılmış su içine 8 ml% 1 tri-sodyum sitrat x 2 H2O ve 160 ul% 1 tannik asit ekleyin. </li><li> 60 ° C&#39;ye kadar çözelti A ve B Isınma ve karıştırılırken karıştırın. Hemen bir koyu mavi renk gözlemleyin. 15 dakika sonra kırmızı bir renk dikkate alınmalıdır. Daha sonra, 95 ° C&#39;ye kadar ısınması 5 dakika tutmak ve oda sıcaklığına kadar çözelti soğutulur. </li><li> Bir karbon kaplı ızgara üzerine NP süspansiyon bir damla ekleyin ve havada kurumaya bırakın. Transmisyon elektron mikroskobu ile NP boyutunu ve morfolojisi kontrol edin. </li></ol> Rodamin N-hidroksisüksinimidil Ester (NHS) ile BSA ve Etiketleme Altın NPlerin 2. Kaplama 16 <ol><li> 30 dakika için 15,000 x g&#39;de, 1.5 ml&#39;lik Eppendorf tüpü içine 900 ul santrifüj altın NP ekleyin. Opduğunda, olabilir bir kerede birden fazla tüpler hazırlamak. </li><li> 900 ul pelet steril Milli-Q su içinde yeniden askıya ve 100 ul, 2 mg / ml BSA / PBS ilave supernatant 30 dakika boyunca 800 rpm&#39;de bir Vortex karıştırın. </li><li> Aşırı BSA kaldırmak için, 60 dakika boyunca 15,000 x g&#39;de santrifüj hazırlanması. </li><li> Süpernatantı atın ve 125 ul PBS altın-BSA NPs yeniden askıya, 1 M bikarbonat 12.5 ul ekleyin. </li><li> Kullanımdan hemen önce, rodamin NHS / DMSO içinde bir 10 mg / ml çözelti hazırlamak altın BSA NPler süspansiyon 1 ml&#39;ye kadar rodamin NHS / DMSO çözeltisi ve 30 ul ekle. Işığa maruz kaçınarak, 800 rpm karıştırma sırasında, oda sıcaklığında 2 saat (ya da O / N) için reaksiyon inkübe edin. </li><li> 48 saat - 36 dönemi boyunca 5 tampon değişiklikleri ile 4 ° C&#39;de PBS karşı diyaliz yoluyla altın BSA-rodamin NPs arındırın. </li><li> , NP stabilize her biri 1 mi altın-BSA-NP rodamin, 200 mg / ml BSA / PBS içinde 10 ul ekleyin. 15 yaşında, ücretsiz veya BSA-rodamin yayımlanan santrifüj kaldırmak için,60 dakika boyunca 000 x g. Işığa maruz kaçınarak, 4 ° C &#39;de OD 520 ve ölçüm haznesine, 2 mg / ml BSA / PBS içinde pelletini. </li><li> Milli-Q veya iki kez damıtılmış su ile NP 10 kez seyreltilir, bir karbon ile kaplanmış ızgara üzerine bir damla ekleyin ve hava içinde kurumaya bırakın. Transmisyon elektron mikroskopisi (TEM) ile NP büyüklüğü ve morfoloji edin. Not: NP hazırlanması için kullanılan bütün malzemeler sterilize edildi ve işlem, bir hücre kültürü kaputu veya alev yanında tezgah üzerinde gerçekleştirilmiştir. </li></ol> HeLa Hücreleri 3. Kültür <ol><li> Sürekli LAMP1-GFP ifade eden HeLa hücreleri% 10 fetal dana serumu (FCS) ile, DMEM ve% 5 CO2 içeren bir atmosferde 37 ° C&#39;de büyütülür. </li><li> 8 oyuklu slaytı (Ibidi) içinde göz başına 50,000 yoğunlukta hücreler Tohum ve / N O inkübe edilir. </li></ol> Bakterilerin 4. Kültür <ol><li> Kullanım Salmonella enterica serovar Typhimurium NCTC 12.023 wilD-tipi (WT) ve soyu. Karşılaştırma için, SIF için anahtar etkeni şifa eksik spi2-T3SS ve Δ şifa içinde Δ ssaV kusurlu mutant soylarda kullanır. Geliştirilmiş GFP yapısal ekspresyon için plazmid pFPV25.1 barındıran suşları kullanın. Not: Bakteriyel suşlar 50 ug / ml karbenisilin ve / veya 50 ug / ml kanamisin (Δ ssaV, Δ şifa ilavesi ile 50 ug / ml karbenisilin (WT) ve LB ilavesi ile (LB) Luria-Bertani suyu rutin olarak kültürlenir ) plazmidleri muhafaza edilmesi için gerekli. </li><li> Sonra taze LB 01:31 sulandırmak ve 3.5 saat büyümesini devam uygun antibiyotiklerle 3 ml LB bakteri tek bir koloni aşılamak ve havalandırma için sallayarak koşullar altında 37 ºC / N O büyür. Bu zaman noktasında, kültürler geç log fazına ulaştıktan ve bakteri, yüksek oranda istilacı bulunmaktadır. Bir &#39;silindir davul&#39; havalandırma test tüpü kültürleri inkübe uygundur. </li></ol> HeLa hücrelerinin 5. enfeksiyon ileSalmonella ve Altın-BSA-Rodamin NPS Chase-etiketleme (şema için Şekil 1&#39;e bakınız) Darbe <ol><li> Alt-kültürlenmiş bakteri OD 600 ölçün ve 1 ml PBS (~ 3 x 10 8 hücre / ml) içinde 600 = 0.2 OD, seyreltik bir çok sayıda elde etmek için 8-çukurlu oda slaytlar HeLa hücrelerinde bakterilerin uygun miktarlarda eklemek enfeksiyon 100 (İB). </li><li> Olmayan içsel bakterileri çıkartmak için 3 kez PBS ile hücre kuluçka makinesi içinde, 30 dakika boyunca inkübe yıkama (bu zaman noktası, 0 saat sonra enfeksiyon ya da 0 saat pi olarak tespit edilmiştir). 100 ug / ml gentamisin içeren, taze kültür ortamının 300 ul ilave edin ve 1 saat boyunca muhafaza edilir. Daha sonra inkübasyon süresi sonuna kadar 10 ug / ml gentamisin ihtiva eden taze ortam ile orta yerine. </li><li> 1 saat süre ile, 100 ug / ml gentamisin ihtiva eden kültür ortamı ile inkübe edildikten sonra, ortam ko (30 mM HEPES, pH 7.4 ile, FCS, L-glutamin, fenol kırmızı ve sodyum bikarbonat olmadan Eagles MEM) orta görüntüleme değiştirilir10 ug / ml gentamisin ntaining. Altın, BSA-rodamin NPler OD 520 = 0.1 nihai bir konsantrasyon elde etmek için, HeLa hücrelerine ilave edilir. Not: Altın-BSA-rodamin NPler da enfeksiyondan önce veya farklı zaman noktaları pi hücrelere ilave edilebilir </li><li> 1 saat inkübe edildikten sonra, ortam maddesinin ayrılması 3 defa PBS ile yıkayın ve inkübasyon süresi içinde geri kalan% 10 FCS ve 10 | ig / ml gentamisin ihtiva eden taze bir görüntüleme ortamında 300 ul ilave edin. Not: kuluçka süresi kullanılan NP ve hücre çizgileri konsantrasyonuna bağlı olarak değişebilir. RAW264.7 makrofaj için, yeterli içselleştirme izin OD 520 = 0.05 bir konsantrasyonda NP o 30 dakika inkübasyon görülmektedir. </li></ol> 6. Görüntüleme <ol><li> Gibi farklı zaman noktalarında yüksek çözünürlüklü görüntüleme için nemlendirilmiş bir ortam odası ile konfokal lazer tarama (CLSM) veya döner diskli (SD) mikroskop gibi bir konfokal görüntüleme sistemi kullanın. </li><li> Sıcaklık c Anahtarıistikrarlı kadar ve sistem ontrol bekleyin. Böyle büyütme, tarama hızı, çözünürlük, Z-yığını vb GFP için uygun uyarma / emisyon ayarlarını kullanın ve altın-BSA-rodamin NP gibi görüntüleme ayarlarını optimize eder. Bu protokol için, 512 x 512 piksel 400 Hz tarama hızı, çözünürlük ve 0.25 mikron Z-adım boyutunu kullanın. GFP ve sırasıyla Ar, lazer (488 nm) ve HeNe lazer (543 nm) kullanılarak altın BSA rodamin harekete geçirir. Hücrelerin şeklini gözlemlemek için parlak alan (BF) kanalı ekleyin. Mikroskopi sistemleri ve enfeksiyon koşulları diğer için, ayar buna göre ayarlanması gerekir. NOT: Bir fotoğraf çarpanı (PMT) detektörü ile tespit edildi BF kanalı ve sinyal ışık kaynağı aynı Ar lazer kullanın. </li><li> Belirtilen zaman noktaları pi anda, mikroskop sahne ve kayıt görüntülerinde enfekte hücreleri içeren 8-iyi odasının slaytlar monte edin. </li></ol> Görüntüler 7. Analizi <ol><li> Mikroskopi görüntü analizi yazılımı kullanın (bkz <strong&gt; Malzeme ve Ekipman Masa) hücre içi Salmonella tarafından Endo-lizozomal sistemin biçimlenme analiz etmek. &#39;Aç&#39; tıklayarak ve dosyayı seçerek verileri açın. NOT: Alternatif olarak, ImageJ veya FIJI gibi açık kaynak yazılım paketleri kantitatif görüntü kullanılabilir analizleri. </li><li> Simgesine görünümü Aşan tıklama nesneleri araç çubuğunda <img alt="Ikon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> Yeni bir yüzey öğesi eklemek. Tıklayın <img alt="Ikon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Sonraki). </li><li> İlgi (ROI) bir bölge seçin segmenti bir ROI analiz etmek. Bir görüntüleme alanında, resmi üzerine bindirilmiş bir dikdörtgen çerçeveli bölüm yatırım getirisi temsil ediyor. Y, ilgili x- değerleri girin, ve z alanlar, ya da doğrudan boyutu ve ROI konumunu değiştirmek için önizleme dikdörtgenin içinde okları tıklatın. Ardından tıklayın <img alt="Ikon" fo:content-width=".1in" src<html> = &quot;/ Files / ftp_upload / 52.058 / 52058icon2.jpg&quot; /&gt; (Sonraki). </li><li> Bir kaynak kanalı olarak seçin Kanal 3 (Altın-BSA-Rodamin NPlerin için sinyali). Elde edilen alanın düzgünlüğü kurmak için yumuşak seçeneği işaretleyin. Elle bir değer tanımlayın veya otomatik olarak oluşturulan değeri kabul. Eşiği için, Mutlak Yoğunluk seçeneğini seçin. </li><li> Eşik ayarı için, manuel seçeneği seçin ve bir değer ayarlayın. Görüntüleme alanında, bir yüzey eşik önizleme gri görüntülenir. </li><li> Sekmesinde sınıflandırır Yüzeyler üzerinde ortaya çıkan yüzeyi çeşitli kriterlere göre filtre edilebilir. Bir varsayılan filtre ve diğer filtreler &#39;voksellerin&gt; 10 sayısı&#39; da &#39;eklemek&#39; tıklayarak dahil edilebilir. Bir filtreleme gerekli değilse, o zaman silme butonuna tıklayarak tüm filtreleri silin. Tıklayın <img alt="Ikon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Sonraki). </li><li> Yüzey oluşturma işlemini tamamlamak için, tıklayın/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt; (Finish). Nesne listesinde, şimdi kontrol un öğe Hacmi ve yeni oluşturulan yüzeyin için kutuyu görüş alanı görüntülenir. </li><li> Istatistik verin. Şimdi, Aşan görünümünde konuların renk alanlarına göre mor kırmızıya değişir. Ve &#39;Plot Numaraları Alan&#39; tablo istatistik bilgilerini (örneğin, kimlik numarası ve nesnelerin alan) gösterir. Tıklama ile bir Excel dosyasına yüzeyin 1 tüm istatistikleri İhracat <img alt="Ikon" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Kaydet). </li></ol>

<ol>
	<li>Coto-Garcia, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+as+fluorescent+labels+for+optical+imaging+and+sensing+in+genomics+and+proteomics.">Nanoparticles as fluorescent labels for optical imaging and sensing in genomics and proteomics.</a> Anal. Bioanal. Chem. 399, 29-42 (2011).</li><li>Xie, J., Lee, S., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+theranostic+agents.">Nanoparticle-based theranostic agents.</a> Adv. Drug Deliv. Rev. 62, 1064-1079 (2010).</li><li>Ruedas-Rama, M. J., Walters, J. D., Orte, A., Hall, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+nanoparticles+for+intracellular+sensing:+a+review.">Fluorescent nanoparticles for intracellular sensing: a review.</a> Anal. Chim. Acta. 751, 1-23 (2012).</li><li>Wu, C., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+fluorescent+semiconducting+polymer+dots+for+biology+and+medicine.">Highly fluorescent semiconducting polymer dots for biology and medicine.</a> Angew. Chem. Int. Ed. Engl. 52, 3086-3109 (2013).</li><li>Giepmans, B. N., Deerinck, T. J., Smarr, B. L., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+light+and+electron+microscopic+imaging+of+multiple+endogenous+proteins+using+Quantum+dots.">Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots.</a> Nat. Methods. 2, 743-749 (2005).</li><li>Kumar, D., Saini, N., Jain, N., Sareen, R., Pandit, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles:+an+era+in+bionanotechnology.">Gold nanoparticles: an era in bionanotechnology.</a> Expert Opin. Drug Deliv. 10, 397-409 (2013).</li><li>Dykman, L. A., Khlebtsov, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+engineered+gold+nanoparticles+into+mammalian+cells.">Uptake of engineered gold nanoparticles into mammalian cells.</a> Chem. Rev. 114, 1258-1288 (2014).</li><li>Chithrani, B. D., Ghazani, A. A., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+size+and+shape+dependence+of+gold+nanoparticle+uptake+into+mammalian+cells.">Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells.</a> Nano Lett. 6, 662-668 (2006).</li><li>Finlay, B. B., Cossart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploitation+of+mammalian+host+cell+functions+by+bacterial+pathogens.">Exploitation of mammalian host cell functions by bacterial pathogens.</a> Science. 276, 718-725 (1997).</li><li>Bhavsar, A. P., Guttman, J. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+host-cell+pathways+by+bacterial+pathogens.">Manipulation of host-cell pathways by bacterial pathogens.</a> Nature. 449, 827-834 (2007).</li><li>Malik-Kale, P., 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=Salmonella+-+at+home+in+the+host+cell.">Salmonella - at home in the host cell.</a> Front. Microbiol. 2, 125 (2011).</li><li>Rajashekar, R., Liebl, D., Seitz, A., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+remodeling+of+the+endosomal+system+during+formation+of+Salmonella-induced+filaments+by+intracellular+Salmonella+enterica.">Dynamic remodeling of the endosomal system during formation of Salmonella-induced filaments by intracellular Salmonella enterica.</a> Traffic. 9, 2100-2116 (2008).</li><li>Schroeder, N., Mota, L. J., Meresse, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella-induced+tubular+networks.">Salmonella-induced tubular networks.</a> Trends Microbiol. 19, 268-277 (2011).</li><li>Drecktrah, D., Knodler, L. A., Howe, D., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+trafficking+is+defined+by+continuous+dynamic+interactions+with+the+endolysosomal+system.">Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system.</a> Traffic. 8, 212-225 (2007).</li><li>Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+preparing+gold+probes+for+multiple-labeling+cytochemistry.">A new method of preparing gold probes for multiple-labeling cytochemistry.</a> Eur. J. Cell Biol. 38, 87-93 (1985).</li><li>Zhang, Y., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+nanoparticles+as+endocytic+tracers+in+cellular+microbiology.">Evaluation of nanoparticles as endocytic tracers in cellular microbiology.</a> Nanoscale. 5, 9296-9309 (2013).</li><li>Pollard, T. D., Earnshaw, W. C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+22.">Chapter 22.</a> Cell Biology. , (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> LysoTracker and LysoSensor Probes. , (2013).</li><li>Shi, H., He, X., Yuan, Y., Wang, K., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+biocompatible+and+long-life+marker+for+lysosome+labeling+and+tracking.">Nanoparticle-based biocompatible and long-life marker for lysosome labeling and tracking.</a> Anal. Chem. 82, 2213-2220 (2010).</li><li>Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+encoding+putative+effector+proteins+of+the+type+III+secretion+system+of+Salmonella+pathogenicity+island+2+are+required+for+bacterial+virulence+and+proliferation+in+macrophages.">Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.</a> Mol. Microbiol. 30, 163-174 (1998).</li><li>Beuzon, C. 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=Salmonella+maintains+the+integrity+of+its+intracellular+vacuole+through+the+action+of+SifA.">Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.</a> EMBO J. 19, 3235-3249 (2000).</li></ol>

No conflicts of interest declared.

memeli hücrelerinin bir endo-lızozomal sistemi besin alımı, hormon-kaynaklı sinyal iletimi, immün gözetim ve antijen sunumu 17 de dahil olmak üzere önemli fizyolojik işlemleri kontrol eder. Şimdi, belirteçlerin çeşitli endositik yolu ve izleme çalışmaları etiketlenmesi için kullanılmıştır kadar. Örneğin, Lysotracker probları seçici düşük iç pH hücre bölmesi içinde birikir ve etkin bir şekilde, nanomolar konsantrasyonlarda 18 de canlı hücreleri etiketlemek olabil...

Vb metal NPlerin, kuantum noktalar, polimer NPlerin, silika NP, karbon noktalar da dahil olmak üzere Floresan nanopartiküller (NPS), geçmiş yıllarda 1,2 sırasında büyük ilgi çekmiştir. Geleneksel organik boyalara kıyasla, floresan NPler gibi güçlü sinyal gücü, direnç photobleaching ve yüksek biyouyumluluk 3,4 olarak arzu kimyasal, optik ve mekanik özellikleri göstermektedir. Bu avantajlar, onları hücre içi algılama ve canlı hücre görüntüleme için tercih edilen yöntem yapmak. Ayrıca, elektron-yoğun NP çeşitli EM 5 ile ultrastrüktürel düzeyde ışık mikroskobu (LM) ve daha yüksek çözünürlükte izleme canlı hücrenin kombinasyonu tanır ilişkili mikroskopik analiz, onların kullanımını kolaylaştıran, elektron mikroskobu (EM) ile görebilir. Örneğin, altın NPler verimli bir şekilde hassas teşhisi için canlı hücreler hem de bağışıklık etiketleme 6 alanında biyosensörler olarak kullanılan uzun zaman olmuştur. Son studies farklı boyut ve şekle sahip, altın NPler hücre kuşaklarının büyük bir çeşitliliği tarafından alımı ve rutin olarak endozomal yolu ile taşıma, dolayısıyla büyük bir potansiyele olmak hücre içi vezikül taşıma takip ve endo-lızozomal sistem etiketleme uygulanmış kolaylıkla olabilir göstermektedir 7,8 . Salmonella enterica, Shigella flexneri ve Listeria monocytogenes gibi Mikrobiyal patojenler, fagositik olmayan konak hücreleri 9 istila farklı mekanizmalar geliştirmişlerdir. Içselleştirilmiş sonra, patojenler, ya sitoplazmada lokalize veya zar-bağlı bölümlerinde tecrit, ev sahibi ortamları ile yoğun etkileşim ve kendi yaşam 10 lehine bu modüle. Örneğin, Salmonella enterica bulunduğu ve bir hücre içi phagosomal bölmesi enfeksiyonu 11 üzerine Salmonella ihtiva-eden çarpıtma (SCV) olarak adlandırılan içinde çoğaltır. olgunlaşan SCV, Golgi aygıtının doğru trafikleri endositik yolunun sürekli etkileşimleri geçiren ve Salmonella -kaynaklı filamentler olarak geniş tübüler yapılar, (SIF), neksin tübülleri sıralama, Salmonella indüklenen salgı taşıyıcı membran proteini 3 (SCAMP3) tübüller, vb oluşumunu uyarır . 12-14. Bu bakteriyel patojenler, konukçu hücre yolları değiştirme konusunda incelenmesi anlama bulaşıcı hastalık için gereklidir. Burada, altın BSA-rodamin NPler konak hücresel endo-lizozomal sistemi etiketlemek için sıvı izleyiciler olarak kullanıldı, ve insan gastrointestinal patojen Salmonella enterica serovar Typhimurium (Salmonella) ile patojen etkileşimlerini incelemek için bir model bakteri olarak kullanıldı endositik yolu ev sahipliği. WT Salmonella ya da mutant suşları ile enfekte enfekte olmayan hücreler ve hücre içi altın BSA rodamin NPler konfokal lazer tarama mikroskobu (CLSM) ile görüntülendi.Daha sonra Imaris yazılım Salmonella enfeksiyonu endozomlar / lizozomların aşırı yeniden düzenlenmesine neden olduğunu gösteren, NP dağılımını ölçmek için kullanıldı. Bu yöntemin açıklaması ardından, benzer deneyler içselleştirilmiş NP uzun vadeli kaderini izlemek ve ökaryotik hücrelerin endositik yolu üzerinde çeşitli eksojen maddelerin veya endojen faktörlerin etkisini araştırmak için dizayn edilebilir.

<table border="0" cellpadding="0" cellspacing="0" width="684">
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			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
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application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

Arzu kimyasal, optik ve mekanik özelliklere sahip Floresan nanopartiküller (NPS) hücre içi organellere etiketlemek için umut verici araçlardır. Burada, biz ökaryotik hücrelerin Endo-lizozomal sistem etiket ve hücre içi patojen Salmonella enterica konak hücresel yolların manipülasyonları izlemek için altın BSA-rodamin NPs kullanarak bir yöntemi tanıtmak. NPS kolayca geç endozomlar / lizozomlarında HeLa hücreleri tarafından içselleştirilmiş ve lokalize edildi. Salmonella enfeksiyonu Salmonella kaynaklı zar yapılarında vezikül ve NP birikimi yeniden düzenlenmesini bağlı. Biz konfokal mikroskopi görüntüleri kantitatif analizleri için Imaris yazılım paketi dağıtmış. nesne sayısı ve enfekte olmayan hücreler içinde boyut dağılımı çok WT Salmonella endo-lızozomal sistemin yeniden modelleme gösteren Salmonella ile enfekte olan hücrelerde olanlardan çok farklıdırlar.

Altın NPler sitrat ve tanik asit chloroauric asidin indirgenmesi ile iyi bilinen bir yöntem ile üretilmiştir. Şekil 2A&#39;da gösterildiği gibi, sentezlenmiş altın NPler yaklaşık 10 nm arasında bir boyuta sahip şeklinde yarı-küresel edildi. BSA-kaplama ve rodamin-etiketleme kendi morfoloji veya boyutunu (Şekil 2B) etkilememiştir. Bu altın NPler hali hazırda çeşitli memeli hücreler tarafından alınır ve endositik sistemleri 7</sup...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Bu makalede, sentezi ve nanopartiküllerin floresan etiketleme (UP) için yöntemler tarif etmektedir. NPler ökaryotik hücrelerin endo-lızozomal sistemi etiket darbe kovalayıcı deneyler uygulanmıştır. Hücre içi patojen Salmonella enterica faaliyetleri ile Endo-lizozomal sistemin Manipülasyon canlı hücre görüntüleme ve sayısal izledi.

Floresan Nanopartiküller Uygulama Hücre içi Bakteri tarafından Endo-lizozomal Sistemi Modelleme Eğitim için

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K Görüntüleme.  Universität Osnabrück.  Bu makalede, sentezi ve nanopartiküllerin floresan etiketleme (UP) için yöntemler tarif etmektedir. NPler ökaryotik hücrelerin endo-lızozomal sistemi etiket darbe kovalayıcı deneyler uygulanmıştır. Hücre içi patojen Salmonella enterica faaliyetleri ile Endo-lizozomal sistemin Manipülasyon canlı hücre görüntüleme ve sayısal izledi. 

Video: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Oligonükleotid çiftli Floresan Mikroküreler kullanarak Kompleksi Klinik ve Çevre Örneklerinde Bakterilerin Multiplex Algılama

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

İmmünoloji ve Enfeksiyon

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Sıcak Sulu-fenol Ekstraksiyon tarafından Gram-negatif bakteriler arınma ve Lipopolisakkaritlerinin Görselleştirmesi

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Vakuolar Kopma Tek Hücre ölçümleri Hücre içi patojenlerin neden

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Bir uyarılabilir İfade Sistemi uygulamak Hücre içi Sinyali ile Bakteriyel Virulans Faktörler Girişim Eğitim için

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

&quot;Acufection&quot; ve Cilt Araştırmaları onun Uygulaması tarafından bir Ekonomik DNA Yerleştirme Sisteminin Geliştirilmesi

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Hücre içi Hedefleme İnhibitörleri Etkinliği ve Sitotoksisite Taraması için sistem<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Çoğaltılmış floresan immunohistokimyasal boyama, görüntüleme ve analiz lenfoma histolojik örneklerinde

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Yapay Olarak Yetiştirilen Bakteriler Tarafından Murin Vajinal Kolonizasyon Modelleri

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...