TB Kistik Fibrozis Kanada araştırma bursu kabul eder.

Araştırma Etik Kurulu (REB) toplamak ve insan denekler gelen balgam örnekleri saklamak için gerekli olduğunu unutmayın. Bu çalışmalar Sick Children REB # 1000019444 için Hastanesi tarafından kabul edildi. 1. Hazırlama CF Balgam Örnekleri <ol><li> kistik fibroz kliniğine rutin ziyaretler sırasında hastaların balgam örneği toplayın ve buz üzerinde tutmak. </li><li> işleme geçmesi, araştırma laboratuarına, koleksiyonun ilk saat içinde buz üzerinde balgam örneği taşıyın. </li></ol> 2. Balgam İşleme <ol><li> Elde edilen balgam örneğinin hacmini kaydedin. Örnek (örneğin, 2 parça PBS, 1 kısım örnek) hacminin 2x fosfat tamponlu tuzlu su (PBS) ekleyin. </li><li> Bir transfer pipet yardımıyla iyice örnek karıştırın. Vortex 1 dk tamamen karıştırmak için en yüksek ayarda örnek. </li><li> Kısım uygun sayı, 1.5 ml mikrosantrifüj tüpler içine yukarıdaki karışımın 1 ml ve 5.000 xg aşağı doğru döndürün4 ° C&#39;de 20 dakika. </li><li> santrifüj sonrasında, süpernatant kaldırmak ve pelet atın. </li><li> Filtre 0.22 um&#39;lik bir filtreden süpernatantı steril ve temiz bir mikrosantrfuj tübüne birikir. NOT: süzüntü sterilite LB agar kaplama ve sıvı ortamları aşılayarak test edilir. </li><li> ileride kullanmak üzere, -80 ° C&#39;de, balgam süpernatant saklayın. NOT: Balgam birden hastalar da filtrasyon aşağıdaki havuza edilebilir dan. </li><li> Balgam Süzüntü, 1/10 hac / hac istenen medyada (balgam 100 ul, ortam 900 ul) seyreltilmiş, kullanılmadan önce. NOT: Burada, standart lizojeni suyu (LB) ortamı kullanılmıştır. </li></ol> Biyofilm Oluşumunun 3. odacıklı coverglass Yöntemi <ol><li> (200 rpm) çalkalanarak 37 ° C&#39;de arzu edilen ortam ilgi gece boyunca bakteriyel izolatı büyütün. Not: Farklı bakteri çok sayıda kullanılmış, Pseudomonas aeruginosa, Staphylococcus aureus klinik izolatlarının dahil,Burkholderia cepacia karmaşık ve Achromobacter xylosoxidans. ortam seçimi Ancak LB ortamı ilk uygulamalar için kullanılabilir suşları ve ilgi koşullara bağlıdır. </li><li> Gecelik kültürden taze ortam 4 ml kültür, 40 ul koyun ve yaklaşık olarak 0.5, 600 nm (OD 600) de optik yoğunluğu ile bir kültür elde etmek için (200 rpm) çalkalanarak 37 ° C&#39;de 3-4 saat boyunca büyümeye -0.6. </li><li> % 10 balgam süzüntü ya da (kontrol olarak) balgam filtrattan olmadan istenilen medyada 1/5 adım 3.2 kültürünü sulandırmak. Balgam filtre bulunan konsantrasyonu test edilebilir (örneğin,% 50 ya da% 100). </li><li> slayt odaları kuyu tohum seyreltme 200 ul kullanın. </li><li> Bakteriler çalkalamadan 37 ° C&#39;de 4 saat eklemesini sağlar. </li><li> 4 saat sonra, medyayı çıkarın ve yavaşça 1x taze medya ile biyofilm yıkayın. 200 ul taze medya ile değiştirin. NOT: biyofilm üzerindeki fres balgam etkilerini araştırmakh medya balgam süpernatant içermelidir. </li><li> biyofilm mikroskopi için saatine kadar yıkama olmadan Ortam her 12 saat yerine, sallayarak olmadan 37 ° C&#39;de zaman istenilen miktarda büyümeye olanak sağlar. NOT: biyofilm antibiyotik duyarlılık balgam Süpernatantları etkisini incelemek için, antibiyotikler biyofilm büyüme 24 saat aşağıdaki ortama ilave edilir ve boyama ve biyofilm görüntüleme kadar medyada muhafaza edilir. </li></ol> 4. Boyama Biyofilmler ve Konfokal Mikroskop <ol><li> İstenilen büyüme süresi (24-48 saat en iyi şekilde çalışır) ardından, oda kuyulardan ortamını çıkarın ve yavaşça steril PBS 300 ul ile iki kez her odasına yıkayın. </li><li> gerekli çözeltinin her ml (canlılık kitte sağlanan), her boyanın 1 ul karıştırılarak biyofilm boyama karışım hazırlayın. Su veya ortam çözelti içinde boya sağlayın. NOT: Su üretici tarafından tavsiye edilmektedir. </li><li> bölmeli covergla her bir kuyu için boya karışımının 200 ul eklep, 45 dakika süreyle karanlıkta, oda sıcaklığında inkübe edin ve. </li><li> odalarından boyama karışımı çıkarın ve steril PBS 300 ul her bir yıkama. PBS çıkarın ve tatlı su ya da medya ile değiştirin. </li><li> konfokal mikroskobu ile biyofilm görselleştirme ile devam edin. </li></ol> 5. Konfokal Mikroskop ile biyofilm görselleştirme <ol><li> hemen (1 saat içinde) boyandıktan sonra odalarına lekeli biyofilm okuyun. Bir seferde 2 8-kuyu odalarına 1 boyanarak slaytların görselleştirme gecikmeyi en aza indirmek. </li><li> edinimi için uyarma ve filtre setleri için lazerler ile konfokal mikroskop kullanılarak görüntüleme gerçekleştirin. NOT: Burada, spektral borealis lazerler ile dönen disk konfokal sistemi (Yeşil: 491nm, Kırmızı: 561 nm) uyarma için kullanılmıştır. 515/40 ve 624/40 emisyon filtre setleri biyofilm canlılığı kiti lekeleri görselleştirmek için kullanılmıştır. </li><li> kamera ile konfokal mikroskop bir 25X su objektif kullanarak fotoğraf çekmek. </li><li&gt; Kullan Z-Yığınları biyofilm modeli. Her 0.5-1 mikron biyofilm son olarak odak çerçeveye ilk odak düzlemi başlayarak görüntü almak (genellikle 30-80 mikron 48 saat biyofilm kapsayan) </li><li> her bir kuyunun 3-5 görüntüleri çekmek. NOT: Böylece 8 de odacıklı coverglass için, 24-40 görüntüleri oluşturulur. </li><li> Analiz için görüntüleri kaydedin. NOT: OME-TIFF dosyalarını COMSTAT 18,19 kullanılarak analiz edilecek şekilde Görüntüler kaydedilmesi gerekir. biyofilm görüntü analizi için talimatlar http://www.comstat.dk/ adresinde bulunabilir. görüntüleri ithal edildikten sonra, bu tür her kanala (kırmızı ve yeşil) için ortalama kalınlığı, biyokütle ve yüzey kapsama gibi parametreler analiz edilebilir. </li></ol>

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms.">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario,+Canada.">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li><li>Vidya, 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=Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis.">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li><li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells.">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li><li>Beaudoin, T., 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+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa.">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li><li>LaFayette, S. 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Bu tarifnamede tarif edilen yöntemler eksojen ürünlerin mevcudiyetinde büyütülen bakteri biyofilmlerin görselleştirme için izin verir. bu tip bir sistem kullanılıyorsa Beklendiği gibi, exoproducts üretimi önem taşımaktadır. Örneğin, Ditiyotreitol (DTT), genellikle örnekleri sıvılaştırılması yardımcı insan balgam kullanılır. Bununla birlikte, tek başına DTT etkisi biyofilm gelişimi ve canlılığı azaltılmış olur (veriler gösterilmemiştir). Bu durumda, tüm koşullar için uygun kon...

Bakteri biyofilmleri birbirlerine bağlanmış ve bir öz salgılanan matris kaplı olan mikroorganizmaların gruplarıdır. 1,2 Klasik olarak, fiziksel olarak akış koşulları altında meydana gelen bir abiyotik veya biyotik yüzeye bağlı bakteri temsil etmektedir. Biyofilmler, aynı zamanda bu tür test tüplerinde oluşan ısı havuzları veya ince zarlar hava-sıvı arayüzü itibarıyla yüzeylerin statik koşullar altında (akış olmaması) ve distal, büyümeye gösterilmiştir. Onlar biyolojik kirlenme, paslanma ve tıkanmalara neden su rezervuarları veya borularda oluşabilir gibi bu biyofilm uzun, endüstriyel prosesler için büyük bir kayba ortamında tanınan ve vardır oylandı. 3,4 Onlar kateter ile ilişkili enfeksiyonlar, kistik fibroz hastalarında akciğer enfeksiyonları, yanı sıra çok sayıda diğer enfeksiyonlarda rol gösterilmiştir olarak biyofilm, aynı zamanda sağlık ortamlarında kritik önem taşımaktadır. Biyofilm enfeksiyonlarının işaretlerinden 5,6 biri de olanBakterilerin antibiyotiklere duyarlılığının katlanmış ve doğuştan gelen bağışıklık sistemi tarafından açıklığı engelli. Biyofilm tabanlı enfeksiyonu kapsayan 7-9 en iyi okudu, klinik olarak anlamlı senaryolar kronik Pseudomonas aeruginosa biyofilm bulaşmış kistik fibrozis (KF) olan hastalarda ortaya çıkar. P. aeruginosa çok zor tedavi yapmak, kronik enfeksiyon kurulması sırasında bir dizi değişiklik uğrayabilir. 10,11 Biyofilmler farklı olarak doğuştan gelen bağışıklık etkinleştirmek ve inflamasyonu sürebilirim. Bu enfeksiyonlar KF hastalarında artmış morbidite ve mortalite neden 12-14 gibi, bu bağlamda biyofilm gelişimini etkileyen faktörleri anlamak önemlidir. Yeni yapılan bir çalışmada konak faktörleri P. aeruginosa biyofilm agrega oluşumunda kritik olduğunu göstermektedir. 15 Bu biyofilm antibiyotik ve konak savunma mekanizmaları indirgenmiş duyarlılık katkıda bulunur. preseörneğin nötrofil elastaz gibi CF akciğer mikroorganizmaların salgılanan ürünler olarak ev sahibi türevli faktörlere nce, büyük ölçüde biyofilm oluşumunu ve gelişimini modüle potansiyeline sahiptir. 16 Ayrıca, biyofilm sayısız yolların ifadesini modüle ve inflamasyonu başlatmak için ev sahibi ile etkileşim. Böyle standart kristal viyole deneyi olarak yüksek kapasiteli yöntemleri, biyofilm süreci ile ilgili olarak bazı bilgiler sağlayabilir iken, bu faktörlere yanıt olarak biyofilm görselleştirme daha derinlemesine bilgi sağlar. Bu yazıda in vitro biyofilm gelişimini incelemek için KF&#39;li hastaların balgam faktörlerini kullanmak için bir yöntem açıklanmaktadır. Bu yöntem, bir ticari biyofilm canlılığı kiti kullanılarak balgam içeren konak faktörlerine maruz kalan biyofilm hızlı görselleştirme sağlar. Bu teknik, görsel exogeno mevcudiyetinde biyofilm büyüme sırasında oluşan değişiklikleri tespit etmek için kullanılabilirUS ürünleri ve çeşitli koşullar altında biyofilm gelişme değişiklikleri analiz etmek için geliştirilmiş bir yöntemi temsil eder.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

Biyofilmler kendinden salgılanan matris kaplı bakteri gruplarından oluşur. Bu endüstriyel kirlenme hem de bir çok sağlık ile ilgili enfeksiyonların gelişmekte ve önemli bir rol oynamaktadır. insan hastalığında en iyi tarif ve çalışılan biyofilm biri kistik fibroz hastalarının kronik akciğer enfeksiyonu meydana gelir. konak bağlamında biyofilm öğrenim görürken, birçok faktör biyofilm oluşumunu ve gelişimini etkileyebilir. Ev sahibi faktörleri biyofilm oluşumunu ve gelişimini etkileyen nasıl tespit etmek amacıyla, balgam yüzer şeklinde bir ev sahibi türevli faktör varlığında, biyofilm büyümeye statik odacıklı coverglass yöntemi kullanılmıştır. Bakteri odalarına tohumlanmış ve balgam süzüntüler maruz kalmaktadır. büyüme 48 saat sonra, biyofilm önce konfokal mikroskopi ve analiz için ticari bir biyofilm canlılığı kiti ile boyandı. Görüntü alınmasının ardından, biyofilm özellikleri farklı yazılım platformları kullanarak değerlendirilebilir.Bu yöntem antibiyotikler de dahil olmak üzere, farklı maddelerin varlığında biyofilm büyüme önemli özelliklerini görselleştirmek sağlar.

Deney genel tasarımı, Şekil 1 &#39;de gösterilmiştir. Bu protokolün kullanılması farklı zaman aralıklarında (örneğin, 24, 48 veya 72 saat) için yetiştirilen biyofilm değişiklikleri görselleştirmek için uygun bir yöntem sağlar. Önemli bir şekilde, örneğin, balgam süzüntüler gibi dış sinyaller, biyofilm gelişme değişiklikleri görselleştirmek için ilave edilebilir. Şekil 2&#39;de de görüldüğü gibi,% 10, ba...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Bir odacıklı coverglass Modeli Kullanılarak Biyofilm Kalkınma Sputum Etkilerinin görselleştirme

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Research

JoVE Journal

Immunology and Infection

8.3K Görüntüleme.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Video: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter Bulaşık Biyofilm Oluşumu Testi

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

İmmünoloji ve Enfeksiyon

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8-iyi Odası Slayt in vitro Biyofilm Oluşumu

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Organik Solvent kullanımı olmadan Triklosan Etkileri: RBL-2H3 Mast Hücre Degranülasyon üzerinde kimyasal etkileri değerlendirmek için bir Mikroplaka Testi

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Ana-Hücre RNA sentezi üzerinde Viral Enfeksiyon Etkisi Tedbir tıklayın Kimya Kullanımı

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Akış adlı lökositlerin üzerine Stromal hücreler Etkilerinin İncelenmesi

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10 parçacık bağlı Bet v 1 Allergen Akışı Sitometrik Analizi

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Gerçek zamanlı görüntüleme ve miktar iki aşamalı dolaşım akış sistemi kullanarak mantar biyofilm gelişimi

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

Bir In vitro Batch-kültür modeli ınsan dışkı microbiota üzerinde girişimsel rejimlerin etkilerini tahmin etmek

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

TNBS Aracılı Intestinal Fibrozis Gelişimi ve Rapamisin Inhibitör Etkilerinin Değerlendirilmesi Üzerine Mekanistik Bakış

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...