The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

Floresan ana hücre dizilerinin 1. Üretim <ol><li> / Oyuk 2 x 10 6 hücre, 6-yuvalı plakalar üzerinde gün 1. Plaka 293T hücreleri (ya da daha başka retroviral paketleme çizgisi) ~% 75 konfluent ertesi gün için. Eğer arzu edilirse, her bir retrovirüs için tabak çift kuyu kullanılır. </li><li> 2. Gün aspire hücreleri ve 2 ml ilave taze büyüme ortamı (DMEM +% 10 FBS + 2 mM L-glutamin). Lipofectamine 2000 veya benzeri bir reaktif kullanılarak ve imalatçının talimatları takip 5-8 ug retroviral vektör (GFP ihtiva eden) ve paketleme vektörünün her biri için (örn., PVSVg) ile hücrelerin transfekte. </li><li> 1 ml büyüme ortamı ile orta yerine, daha sonra 37 ° C CO2 inkübatöründe 4-6 st için DNA ile inkübe hücreleri, vb. </li><li> 37 ° C CO2 inkübatöründe 48 saat süreyle inkübe hücreleri. </li><li> 3. Gün Plaka hedef hücreler (örn., HeLa) 10 5 hücre yaklaşık yoğunluğunda 6 oyuklu plakalar üzerine / kuyu olmak ~% 50 konfluentertesi gün. Ters bir mikroskop GFP floresan izleyerek 293T hücrelerinde pozitif transfeksiyon doğrulayın. </li><li> 4. Gün Collect retrovirüs-ihtiva eden bir 0.45 mikron selüloz asetat şırınga filtre ile süpernatan bir pipet kullanılarak ve filtre 293T hücrelerinde rahatsız süpernatantlar. </li><li> HeLa hücreleri Ortamı çıkarın ve 16 ug / ml ihtiva eden 0.5 ml polybrene büyüme ortamı ilave edin. Hücrelere süzüldü retrovirüs ~ 0.5 ml ilave akıllı-damla ve 24 saat boyunca 37 ° C CO2 inkübatöründe inkübe hücreleri. 4 ° C &#39;de (de yinelenen, örn) kalan herhangi bir retrovirüs saklayın. </li><li> Yinelenen kuyuları seri kalan retroviral parçacıklar ile enfekte için ekstra retrovirüs adımı yineleyin 1.7 üretmek için yapılmış ise Günü 5.. </li><li> (Örneğin, 500 ug / ml neomisin) 10 cm&#39;lik bir çanak içeren seçim antibiyotik olarak 1: Gündüz 6. Geçiş HeLa hücreleri 1 transfekte edildi. </li><li> Seçim Antibiyotik varlığında tekrar büyümeye kalıt aktarımlı hücrelerin için yeterli bir süre bekleyinHücreler ayıklama antibiyotiğin daha düşük bir konsantrasyonu ile standart teknikler tarafından yayılan olabilir sonra tlc, (örn., 200 ug / ml neomisin). Not: Gerekirse Hücreler de klonal, şu anda ideal bir parlaklık için seçilmiş olabilir. </li></ol> GFP-ifade Chlamydia trachomatis &#39;in 2. Üretim <ol><li> 2x CaCl2 tamponu (20 mM Tris pH 7.4, 100 mM CaCl2) ile 4SP tamponu (0.4 M sukroz, 16 mM Na 2 HPO 4) hazırlayın. </li><li> Gün 1. çözülme dondurulmuş C. trachomatis stok buz üzerinde ve hemen 50 ul 2x CaCl2 tampon içine 10 ul bakteri sulandırmak. 3 ug plazmid DNA ekleyin (örn.,, PASK-GFP-mKate2-L2), iyice karıştırın ve 25 ° C&#39;de 30 dakika inkübe edilir. </li><li> Bu adım sırasında, bir santrifüj tüpüne McCoy hücreleri, T-75 balon trypsinizing konakçı hücrelerin hazırlanması. 5 dakika boyunca 50 xg&#39;de hücre süspansiyonu santrifüj ve supernatant atın. Resuspen1x CaCI2 tampon uygun hacimde d hücre topağı 4 x 10 7 hücre / ml bir nihai konsantrasyon elde edilmiştir. </li><li> (Adım 2.2) 30 dakika süreyle inkübe edildikten sonra, transformasyon karışımı borusu (toplam hacim 200 ul) ile McCoy hücreleri hazırlandı 100 ul ve 25 ° C&#39;de ek bir 20 dakika inkübe edilir. 2 ml büyüme ortamı ile bir 6-yuvalı plaka ve kaplama tek bir kuyusuna bu dönüştürülmüş hücre karışımı 100 ul ekle. 2 gün bir CO2 inkübatöründe 37 ° C&#39;de inkübe edin. </li><li> 3. Gün Lyse McCoy hücreleri C ile enfekte bükülmüş 1000 ul pipet ucu ile hücreleri, 1 ml dH 2 O ile orta yerine ve kazıyarak trachomatis. C. verimli hücre erimesi ve serbest bırakılması için 5 kez 1 ml 4SP tamponu ekleyin ve ~ 27.5 G iğne ile süspansiyon geçmektedir trachomatis. </li><li> C ihtiva eden hücre lizatı devri 2 mi Taze kaplama MM konfluent tek tabaka ihtiva eden bir T-75 balonuna trachomatisoy hücreleri; (FBS&#39;siz) 8 ml DMEM ekleyebilir ve C izin vermek için 25 ° C&#39;de 2 saat boyunca inkübe trachomatis hücrelerine yapışma. </li><li> Aspire hücreler 10 U / ml penisilin G ve 1 ug / ml siklohegzimid ile desteklenmiş taze büyüme ortamı eklemek. 37 ° C CO2 inkübatöründe 48 saat boyunca balon inkübe edin. </li><li> 4. Gün hücreleri enfekte ve inklüzyonlar anormal Klamidya ihtiva ışık mikroskobu ile doğrulayın. 2&#39;den büyük mikron çapında olan kapanım içinde halka benzeri nesneler gibi standart ışık mikroskobu üzerinde parlak vakuol ve anormal Chlamydia organları olarak kapanımlar tanımlayın. </li><li> Bir süspansiyon içine hücreleri 2 ml dH 2 O ile orta yerine ve kazıma tarafından Gün 5. lizleyin kültürü. 2 ml 4SP tamponu ekleyin ve ~ 27.5 G iğne ile 5 kez süspansiyon geçmektedir. </li><li> Bir 6-çukurlu plaka içindeki tek bir kuyuda McCoy hücreleri taze tek tabaka enfekte hücre lizatı 2 ml kullanın. 25 ° C, aspirat 2 saat süreyle lisatı ile inkübe hücreleri ve taze büyüme eklemePenisilin G ve sikloheksimid içeren ortam. </li><li> Hücrelerin genel sağlığına bağlı olarak 3-5 gün boyunca 37 ° C CO2 inkübatöründe inkübe hücreleri. </li><li> Tekrarlama (anormal cisimlerin yoksun) Normal görünümlü inklüzyonlar ortaya çıkmıştır kadar 6 plaka taze kuyulara 2,9-2,10 bir veya birkaç kez, gerektiğinde, Pasajlanması kültürleri yineleyin. Şu anda kontrol etmek ters bir floresan mikroskop kullanmak C. trachomatis mKate2 (kırmızı) ifade eder. </li><li> T-150 şişelerinde yetişen enfekte McCoy veya HeLa hücrelerinden Chlamydia transformantların hasat örneğin yüksek titre klamidyal stoklarının oluşturulması için enfekte kültürler, büyütmek. </li></ol> 3. Chlamydia ile Hücreler bulaşmasını <ol><li> IFU tespit edilmesi ve çok-yuvalı tarama için, örneğin cam alt yemekler ya da oda slaytlar yüksek çözünürlüklü görüntüleme için, ya da 24 oyuklu plakalar için istenen kültür kabı Plaka GFP-HeLa hücreleri,. </li><li> Fro ÇözülmeC içeren zen tüp trachomatis, C. muridarum veya C örnek İçişleri Bakanlığı 1 = buz üzerinde pneumoniae ve enfeksiyon istenen çokluğu (İçişleri Bakanlığı) için HBSS sulandırmak. </li><li> Kullanılacak belirli Chlamydia zorlanma dayalı statik veya santrifüj destekli ya enfeksiyonları gerçekleştirin. <ol><li> C ile enfeksiyonlarında trachomatis LGV serovar L2 veya C muridarum, HBSS GFP-HeLa hücreleri yıkayın ve 25 ° C&#39;de 2 saat süre ile seyreltilmiş bakteri ile inkübe edin. Aspire edin ve HBSS ile yıkama hücreleri ve 37 ° C CO2 inkübatör büyüme ortamında hücrelerin inkübe edin. </li><li> C. trachomatis serovar D veya C ile enfeksiyonlarında pneumoniae, HBSS GFP-HeLa hücreleri yıkamak ve hücrelerin seyreltilmiş bakteri ekleyin. Bir tezgah üstü santrifüj içinde sallanan bir kova rotor eki bir multiwell plaka tutucu multiwell plaka ya da (bir multiwell plaka kapağında bant ile güvence altına) cam alt çanak yerleştirin. </li><li> 1 saat boyunca 25 ° C&#39;de 900 x g&#39;de santrifüj hücreleri. </li&gt; <li> 1 saat boyunca 37 ° C CO2 inkübatör santrifüj ve yerine kültür damarları çıkarın. </li><li> Aspire hücreleri biyogüvenlik kabini, HBSS ile iki kez hücreleri yıkayın ve taze besi ekleyin. 37 ° C CO2 inkübatör yerleştirin hücreleri. </li></ol></li></ol> Chlamydia İçermelerin 4. Canlı Hücre Görüntüleme <ol><li> CO 2 inkübatör Chlamydia bulaşmış GFP-HeLa hücreleri çıkarın ve fenol + 5 kırmızı% FBS&#39;siz RPMI ile orta yerine. </li><li> 20X ya da daha yüksek yağ objektif kullanarak bir ters flüoresan mikroskop (Nikon Eclipse Ti-E veya benzeri) üzerine monte hücreleri. </li><li> Büyük kara delikler (Klamidya kapanımlar) içeren yeşil hücreler: görüntüleme yazılımı (Volocity 6 veya benzeri) kullanarak, odaklanmak ve tespit Chlamydia GFP-HeLa hücreleri enfekte. </li><li> Mark görüntüleme yazılımı kullanarak ilgi Chlamydia ile enfekte olan hücreleri içeren bölgelerin xyz yerleri (Volocity 6 veya benzeri). El XY Sahne görünüm modunda süre &#39;Puanları ekleyerek&#39; Bu gerçekleştirin. Bu şekilde, xyz koordinatlar her işaretli yeri için kaydedilir. </li><li> Toplama Kurulumu iletişim kutusunda, yeşil (GFP, HeLa sitosol) ve kırmızı (mKate2, C. trachomatis) kanalları ve kanal başına yeni çerçeveler her 5 dk hızında elde etmek yazılımı yapılandırın. </li><li> Protokol &quot;Yakalama / Kaydet&quot; butonuna tıklayarak, yukarıda girilen edinimi kullanarak görüntü dizisi edinin. </li></ol> Canlı hücreler Eklenmesi Büyüme Parametrelerinin 5. kantitasyonu <ol><li> İstenilen zamanlarda enfeksiyon sonrası (örn., 24, 48 hpi) Chlamydia inkübatör GFP-HeLa hücreleri enfekte kaldırmak ve ters floresan mikroskop monte. Not: Bir yüksek NA ile 20X veya 40X hava hedefi 24-kuyucuğu için en uygun olduğunu ve 40X petrol hedefi haznesi slaytlar için tavsiye edilir. Bu eşek için her iki alt tabaka üzerinde hücreleri büyümekay. </li><li> Inklüzyonlar maksimum çapı altındadır hücrelerin Midplanes odaklanın ve net kenarları verim. </li><li> Her bir oyuğa (oyuk başına en az 10) birçok alanları için görüntüler elde; kuyular genellikle çoğaltır veya deneysel değişkenleri temsil etmektedir. </li><li> Gözle (kapanım GFP-HeLa hücrelerinde siyah bölmeleri olan), elle kapanım sayısını numaralandırmak veya otomatik olarak kapanımlar tanımlamak ve saymak için görüntüleme yazılımı algoritmaları kullanır. <ol><li> Floresan yoğunluğu ile GFP-HeLa hücreleri bul (komutunu &#39;Nesneler Bul&#39;) ve hücrelerin göreceli yoğunlukları dayalı floresan eşik yoğunluğunu ayarlayın. </li><li> Bu sadece 5&#39;inden um 2 tutmanın büyüklüğü yönergeleri kullanarak Filtre, seçilen nesneler. Bu &#39;nüfus 1 &quot;dir. </li><li> Girdi olarak &#39;nüfusu 1&#39; kullanarak komuta &#39;Nesneler Holes doldurun&#39; uygulayın. Bu adım &#39;nüfusu 2&#39; oluşturur. </li><li> &#39;S çıkarınız&#39; nüfus 1 &#39;opulation 2 &#39;nüfusu 3&#39; olumlu olarak belirlenmiş Chlamydia kapanım, işaretli ürün olarak vermek üzere &#39;. </li><li> Örnek tutmak nesneler için, &#39;nüfus 3&#39; (&#39;Filtre Nüfus&#39; komutu) boyut filtre uygulamak 24 saat veya daha uzun süre enfekte hücrelerde kapanım için daha büyük 25 mikron 2. Bu kapanımlar çok daha küçük gibi önce 18 saat gibi erken enfeksiyon süreleri için, set boyut filtresi, 4 mikron 2&#39;den büyük nesneleri tutmak için. &#39;Kapanım&#39; olarak adlandırılan nesnelere nüfusu Boyut filtreleme sonuçları. </li><li> Görüntüleme yazılımı kullanarak, kantitatif örneğin görüntü veri, içerme numarası, inklüzyon boyutunu ve içerme çevresi hesaplayın. </li></ol></li></ol>

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Yazarlar hiçbir rakip mali çıkarlarını var olduğunu beyan ederiz.

Burada gerçek zamanlı görselleştirme ve Chlamydia kapanım analizi için floresan konakçı hücreleri üretmek için deneysel bir strateji açıklar. Bu vakuol görselleştirme yaklaşımı zamanla tek hücre hücre popülasyonlarında karşısında ya da klamidya kapanım dinamik özellikleri, aydınlatmak izlemek ve kantitatif ölçmek için güçlü yeteneği kazandırır. Klamidya kapanımlar floresan proteini etiketli hücrelerde çarpıcı iyi kolayca tespit edilir, öyle ki tanımlanır Ek ...

Hücre içi bakteri Chlamydia türlerinin neden Bulaşıcı hastalıklar cinsel yolla bulaşan hastalık, pelvik inflamatuar hastalık, körlük, zatürre ve muhtemelen ateroskleroz 1-4 dahil olmak üzere küresel sağlığı üzerinde önemli bir yük, ortaya. Bir vakuol içinden, konakçı hücre ile etkileşime Chlamydia kabiliyeti (dahil olarak adlandırılır), hücre ve ev sahibi başarılı enfeksiyonu için kritik bir belirleyicidir. Içerme chlamydia büyümesini sağlayan ve dinamik Chlamydia 5 tüm 2-3 gün gelişimsel döngüsü boyunca değiştirilmiş yeni bir patojen bölme olduğunu. Klamidyalar ve zorunlu hücre içi doğası doğrudan dahil eşsiz biyoloji eğitimi için özellikle araştırma topluluğuna sayısız zorluklarla, sunar. Büyük bir handikap verimli hücre içi Klamidya veya floresan yaklaşımla kendi eklenmesi ya görselleştirmek için yetersizlik olmuşturcanlı hücrelerde es. Bir son keşif sonunda GFP C ifade oluşturmak için bir araç ortaya çıkarmıştır trachomatis 6; Ancak bu bulgu henüz dahil belirli etiketlenmesine yol açmamıştır. Bazı teknikler bakteri ve inklüzyonlar 7,8 etiketlenmesi için tarif edilmiştir, fakat bu ışıkla ağartma olmayan spesifite, geçicilik ve duyarlılık gibi bazı eksikleri vardır. Grubumuz tarafından anahtar keşif konakçı hücreleri 9 ifade GFP kullanarak içermenin aydınlatılması için yeni bir strateji kurdu. Bu strateji, rasyonel daha büyük 520 Da 10 molekülüne dahil membran iç sızdırmazlık patlatır. Hücreler stabil belirli bir sitozolik floresan proteini (örneğin, GFP veya mCherry) ifade etmek için tasarlanmıştır zaman Chlamydia inklüzyonlar floresan onların tam dışlama yoluyla kayda değer bir açıklıkla görebilir. Bu ters görüntüleme stratejisi, tüm Chl için kapanım derhal görselleştirme sağlaramydia türleri ve havaalanı en konakçı hücreler için adapte edilebilir. Kendi programını bir göstergesi olarak, bu yöntem Chlamydia spp 9 hücresel çıkış yollarını ortaya çıkarmak ve tanımlamak için önceden kullanıldı. Burada, biz daha fazla bu yöntem gerçekleştirilir ve içerme büyüme dinamikleri hakkında önemli nicel veriler elde etmek için istismar edilebilir gösterilmektedir. Bundan başka, etkili bir şekilde pahalı antikor bazlı sayım yöntemleri yerine kullanılabilir ve bu tür Chlamydia 11 mKate2 ifade eden diğer floresan etiketler, ile birlikte de kullanılabilir. Araçlar Bu güçlü kombinasyon yaşayan konak hücrelerin içinde klamidya içerme zarının fiziksel özelliklerinin araştırılmasını sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

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

Sitosolik floresan proteinleri (örneğin GFP) ifade eden memeli hücrelerinin, canlı, enfeksiyonlu hücre kültürlerinde Chlamydia inklüzyonlar aydınlatma sağlamak için tasarlanabilir. Chlamydia enfeksiyonu üzerine, inklüzyonlar konakçı hücrelerde (Şekil 1) siyah noktalar olarak kolaylıkla görebilir. Floresan eksik kapanım netliği görünümü ve / veya tedaviler (Şekil 1) çok sayıda alanları arasında kapanım otomatik tanımlanması içi...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

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

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

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

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

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

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

7.5K Görüntüleme.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Tümör Hedefli ölçülmesi Büyüme ve Gen İfade Dynamics<em&gt; S. Typhimurium</em&gt; Bakteri

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Ileri Genetik Yaklaşımlar<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membran Kutup, Membran Dürüstlük ve Hücre içi İyon Konsantrasyonlarında İzleme değişiklikler<em&gt; Streptococcus pneumoniae</em&gt; Floresan boyalar kullanarak

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

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

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

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

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

"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.

&quot;Phagosome Closure Assay&quot; 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 ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Hassas kesimli Canlı Pulmoner Dendritik Hücreler Visualize&#39;a Fare Akciğer Dilimleri

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.