Yazarların Dr teşekkür etmek istiyorum. Rick Ryan, Umesh Patel, Jeff Till Matthew Hsu, Jehangir Mistry ve bu projeler ve protokoller geliştirilmesi sırasında destek Michele Hatler.

Cep Hazırlanışı: <ol><li> Büyüme ortamı içinde tahlil, kültür nöronlar / astrositler hücre ekim öncesinde ~% 70-80 kadar konfluent (hücre çözülme veya izolasyon doğrudan ekim sürece). </li><li> Ilgi hücre tipi için uygun yöntemi ile kültür şişeler / plakaları hücreleri ayırın. Gerekirse, poli-D-lizin veya ekstrasellüler matriks protein ile kat tahlil plaka kuyu hücre yapışmasını geliştirmek için. Astrositler ve nöronlar Co-kültürler, aynı anda veya sırayla numaralı seribaşı olabilir. Sıralı turlar için, &quot;bazal&quot; katmanı olarak ilk astrositler kaplama, gerekirse nöronal kaplama ve farklılaşma takip önerilir. , Hücre tipi, daha sonraki kültür, ilgi parametreleri uygun olarak hücre yoğunluğunu ayarlayın vb kültür yaşına bağlı olarak, hücre kaynağı ve ekim yoğunluğu, birincil kültürlerin çoğalması, GFAP ifade veya neurite akıbet oranı büyük ölçüde değişebilir önemlidir karakterize ve deneysel model için en biyolojik önemi yanı sıra, HCS (bkz. Şekil 1) kullanarak etkili görüntüleme, segmentasyon ve analiz sağlamak için sağlamak için hücre sisteminizi optimize. Plaka (hücrelerin toksin tedavisi kolaylaştırmak için, aşağıda Adım 3 açıklandığı gibi 90 mcL medya numaralı seribaşı olabilir) hücreler ekledikten sonra, hatta hücre dağılımı sağlayarak, plaka, 15-30 dakika süreyle oda sıcaklığında düz bir yüzey üzerinde oturmak için izin verir. Bu dönemin ardından, en az 24 saat inkübe hücreler için büyüme ortamı (37 ° C /% 5 CO 2), daha sonra farklılaşma kültür koşullarına geçiş (örneğin, PC12 hücreleri düşük serum / NGF), uygun şekilde. Hücreleri, hücre tipi için uygun aralıklarla değişen medya, izdiham ya da farklılaşma istenilen seviyeye ulaşıncaya kadar kültür devam edin. </li><li> Hücre tedavileri (kontrol bileşikler, test bileşikleri, vb.) Ilgi tedavi süresi kurs uygun olarak, bu kültürün döneminde herhangi bir noktada tanıtıldı olabilir. Akrilamid, hidrojen peroksit ve K-252a, nörotoksik kontrolü bileşikler olarak verilmektedir. Yeterli reaktifler, beş 96-kuyucuğu yinelenen 12-nokta doz cevap eğrileri (doz cevabı içinde bir dH 2 O veya DMSO kontrol seti de dahil olmak üzere) sağlanır. Bileşikler 250X konsantrasyonu (K-252a için, 1 mcM maksimum tedavi varsayarak) veya 10X (akrilamid ve hidrojen peroksit, sırasıyla 100 mM ve 10 mM, maksimum tedavi varsayarak) verilmektedir. Önerilen tedavi hazırlık yarım günlük içerir: (1 √ 10) Bileşik Sulandırma Tamponu seyreltme tarafından takip DMSO 250X bileşik seri seyreltme, 10X 10X kontrolü bileşikler (dH 2 O menşeli seri steril dH 2 O doğrudan seyreltilmiş olabilir veya Bileşik Sulandırma Tamponu). 10 mcL her tedavinin ardından nihai 1X konsantrasyonu (% 0.4 DMSO veya% 10 dH 2 O) için, her bir kuyunun zaten mevcut kültür ortamı 90 mcL eklenebilir . Örnek veri az 37 ° C fiksasyon için önce 24 ya da 96 saat boyunca bileşik tedavi sağlanır. </li></ol> Hücre Fiksasyon ve Immunofluorescent Boyama: Not: Boyama süresi ~ 2.5 saat sonrası fiksasyon. Kuyu boyama adımları arasında kurumasına izin vermeyin. Aspirasyon ve muafiyet reaktifler, sıvı kayma nedeniyle herhangi bir hücreyi kaybını azaltmak için, düşük akış hızlarında yapılmalıdır. Önerilen tüm birimlerinin yanı başına &#39;96-iyi mikroplak iyi tek bir bakın. Hacimleri &#39;96-plaka başına&#39; tüm önerilen sıvı taşıma hacim kaybı% 25 fazla. Tüm boyama adımları oda sıcaklığında (RT) yapılmaktadır. Tüm tamponları ve antikor çözümleri RT en az 24 saat süreyle stabildir. <ol start="4"><li> Kültür dönemi sonunda, oda sıcaklığında (RT) veya 37 º C ön sıcak HCS Fixation Çözüm (2X) istenirse (12 mL/96-well plaka). De, doğrudan / kültür ortamı kimyasal bir davlumbaz, 100 mcL ekleyin ve 30 dakika oda sıcaklığında düzeltmek için izin. (MSDS) fiksatif / toksin içeren medya çıkarın ve tehlikeli atıkların yönetmeliklere uygun olarak bertaraf. Boyamadan hemen geçmeden, HCS İmmünofloresan Tampon 200 mcL ile iki kez iyice durulayın. Alternatif olarak, plakalar daha sonraki bir zamanda boyanmış isteniyorsa, kuyu ve boyama kadar 4 º C&#39;de sıkıca mühürlenmiş plakları ikinci durulama hacmi bırakın, daha sonra iki kez 200 Wash Buffer mcL ile durulayın. </li><li> Boyamadan önce 4 ° C sabit örnekleri saklanan varsa, 200 mcL HCS İmmünofloresan Tampon boyama protokol ile devam etmeden önce iki kez yıkayın. </li><li> Tavşan anti-βIII tubulin / Fare Anti-GFAP HCS Primer Antikorlar (6 mL/96-well plaka) çözümü şu şekilde hazırlayın: 5.88 mL HCS İmmünofloresan Tampon her çözülmüş birincil antikor 60 mcL ekleyin. İyice karıştırın. Önceki İmmünofloresan Tampon durulama çıkarın. İlköğretim Antikor çözüm iyi 50 mcL ekleyin ve oda sıcaklığında 1 saat inkübe edin. </li><li> İlköğretim Antikor çıkarınçözüm. 200 mcL HCS İmmünofloresan Tampon ile üç kez durulayın. </li><li> HCS İkincil Antikor / Hoechst HCS Nükleer Leke (6 mL/96-well plaka) çalışma çözüm aşağıdaki gibi hazırlayın: 30 mcL her çözülmüş ikincil antikor ve çözülmüş Hoechst HCS Nükleer 30 mcL HCS İmmünofloresan Tampon 5.91 mL Leke ekleyin. Çözüm ışıktan korunması, iyice karıştırın. Önceki HCS İmmünofloresan Tampon durulama çıkarın. HCS İkincil Antikor / Hoechst HCS Nükleer Leke çözüm 50 mcL ekleyin ve ışıktan korumalı RT, 1 saat boyunca inkübe edin. </li><li> HCS İkincil Antikor / Hoechst HCS Nükleer Leke çözüm çıkarın. 200 mcL HCS İmmünofloresan Tampon ile iki kez durulayın. </li><li> Önceki HCS İmmünofloresan Tampon durulama çıkarın. Kuyularda ikinci durulama hacmi bırakarak, HCS Yıkama Tamponu 200 mcL ile iki kez durulayın. </li><li> 4 º C&#39;de hemen Seal plaka ve görüntü veya mağaza plaka görüntüleme için hazır olana kadar ışıktan korunmalıdır. </li></ol> Görüntü Alma ve Analiz: <ol start="12"><li> Hücre Analyzer IN (GE Healthcare) dahil olmak üzere mevcut HCS platformlar, ArrayScan (ThermoFisher Bilimsel) veya Opera (Perkin Elmer) çeşitli üzerine lekeli plakaların görüntüleme ve analiz yapılabilir. Görüntüleme ve analiz için bazı kurallar Tablo 1&#39;de verilmiştir: </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222 Görüntü Toplama Kuralları </td></tr><tr><td> Algılama Reaktif </td><td> Objektif </td><td> Uyarıcı filtre Aralığı [pik / bant genişliği (nm)] </td><td> Emisyon Filtre Aralığı [tepe / bant genişliği (nm) ] </td></tr><tr><td> Hoechst HCS Nükleer Leke </td><td> 20X </td><td> 360/40 </td><td> 460/40 (veya gerekirse 535 / 50) </td></tr><tr><td> HCS İkincil antikor FITC-Eşek, anti-tavşan IgG </td><td> 20X </td><td> 480/40 </td><td> 535/50 </td></tr><tr><td> HCS İkincil Antikor Cy3-Eşek, anti-fare IgG </td><td> 20X </td><td> 535/50 </td><td> 600/50 </td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222 Görüntü Analiz Rehberi </td></tr><tr><td> Hücre Parametre </td><td> Bulma </td><td> Segmentasyon / Ölçme </td><td> Gerekçe </td></tr><tr><td> Hücre sayısı, Nükleer Özellikleri </td><td> Hoechst HCS Nükleer Leke </td><td> Nükleer bölge (460 nm emisyon kanal). Çekirdeklerin sayısını sayın. DNA içeriği (nükleer yoğunluğu) ya da nükleer alan analizler de mümkündür. </td><td> ΒIII-tubulin veya GFAP ifade ile ilişkili ayrı nöronal ve astrositik hücre sayımı / karakterizasyonları (şiddetle önerilir) elde etmek için bu çekirdekleri &quot;filtre&quot; hücre sayısı, nükleer özellikleri, hücre kaybı, toksisite fenotipleri, vb belirlemek için kullanın. </td></tr><tr><td> βIII tubulin İfade, Neurite akıbet </td><td> HCS İkincil antikor FITC-konjuge </td><td> Sitoplazmik bölgesi (535 nm emisyon kanal). FITC sinyali neurites (örneğin, asgari / ortalama hücre vücut bölgeleri, minimum / maksimum neurite uzunlukları ve genişlikleri ile) nöronal hücre gövdeleri ayırmak için kullanılan olabilir. Toplam neurite uzunluğu, neurite sayar / hücre, vb gibi parametreleri belirleyin </td><td> Neurite akıbet ölçümler nöronal farklılaşma sırasında veya kimyasal yaralanma, hastalık durumları, vb. Bir sonucu olarak modüle olabilir farklı nöronal karakterizasyonu (şiddetle önerilir) elde etmek için βIII-tubulin ekspresyonu ile ilişkili olanlar için &quot;filtre&quot; hücre gövdeleri. </td></tr><tr><td> GFAP İfade, Astrosit Hücre Alanı </td><td> HCS İkincil Antikor, Cy3-konjuge </td><td> Sitoplazmik bölge (600 nm emisyon kanal). Cy3 sinyal astrositik sitoplazmik segmentasyon tanımlamak için kullanılan olabilir. Ortalama sitoplazmik sinyal yoğunluğu, hücre alanı, vs gibi parametreleri belirleyin </td><td> GFAP ifade ve astrosit hücre alanı astrosit gelişimi sırasında ya da kimyasal yaralanma, hastalık durumlarının bir sonucu olarak modüle olabilir, vb hücre gövdeleri farklı astrositik karakterizasyonu (şiddetle önerilir) elde etmek için GFAP ifade ile ilişkili olanlar için &quot;filtre&quot;. </td></tr></tbody></table> Tablo 1. Resim Alma ve Analiz Yönergeleri - HCS222 βIII-Tubulin/GFAP (Co-Culture) Testi Temsilcisi Sonuçlar: <img src="/files/ftp_upload/1173/figure31173.png" alt="Şekil 3" /> Şekil 1. βIII-tubulin ve GFAP immunofluorekarışık sıçan sinir hücre kültürleri scence. Birincil sıçan hipokampal nöron veya sıçan PC12 hücreleri ya primer sıçan hipokampal astrositler Co-kültürler nöronal tohumlama kültür birkaç gün ön-kaplama astrositler tarafından üretildi. PC12s farklılaşma koşulları (düşük serum / NGF) altında ek bir 2 gün süreyle kültüre iken primer nöronlar, ek bir 11 gün kültüre edildi. Hücre işleme, fiksasyon ve immün HCS222 tahlil protokollere göre yapılmıştır. Hücreler Hücre Analyzer 1000 (3.3) 20X (primer nöronların) veya 10X (PC12) amacı büyütme IN GE görüntülü ve GE IN Hücre Analyzer 1000 İstasyonu (3.4) Neurite üremeyi ve Çoklu Hedef Analizi algoritmaları kullanılarak analiz Üst panel. Hoechst HCS Leke Nükleer (mavi), βIII-tubulin (yeşil) ve GFAP (kırmızı) floresan Sigortalı görüntüler. (Mavi (birincil nöronların hücre gövdeleri özetlendiği gibi) ve sarı (PC12), yeşil (birincil nöronların) ya da açık mavi (PC12) neurites orta panel) βIII tubulin floresan kanalı ayrı ayrı analiz neurite akıbet segmentasyon için izin verir. GFAP kanal analizi astrosit segmentasyon (yeşil çekirdekleri mavi, sitoplazma ana hatlarıyla alt panel) sağlar. Için ayrı hücre sayımı sağlayan, (kırmızı) (-) birincil sıçan hipokampal nöron / astrosit ortak kültür GFAP segmentasyon çekirdekleri / hücre GFAP (+) (yeşil özetliyor) organları ve GFAP olduğunu ayırt etmek yeteneğini gösteriyor nöronlar ve karışık bir kültür ortamda astrositler. <img src="/files/ftp_upload/1173/figure41173.png" alt="Şekil 4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="Şekil 4 part2" /> Şekil 2. Doz yanıtları birincil sıçan hipokampal nöron / toksik streslere karşı ortak kültürler astrosit. İlköğretim sıçan hipokampal astrositler büyüme ortamı (P6) 96-kuyucuğu kaplama ve 6 gün boyunca kültüre edildi. İlköğretim sıçan hipokampal nöron (çözülme doğrudan) daha sonra ön kaplama astrositler üstüne numaralı seribaşı ve 11 gün boyunca büyüme medyada kültüre edildi. Hücreler seri dilüsyonları kültürünün son 24 saat akrilamid ya da hidrojen peroksit (maksimum konsantrasyonları = 100 mM ve 10mM, sırasıyla) ile tedavi edildi. Hücre işleme, fiksasyon ve immün HCS222 tahlil protokollere göre yapılmıştır. Hücreler Hücre Analyzer 20X (10 alanlar / iyi) 1000 (3.3) GE IN görüntülü ve Hücre Analyzer 1000 İstasyonu (3.4) Neurite üremeyi ve Çoklu Hedef Analizi algoritmaları GE IN (sitoplazmik / / nükleer neurite segmentasyon) analiz edildi. Sunulan veriler ortalama ± SEM, n = 3. Çoklu parametreleri (neurite akıbet, GFAP yoğunluğu, astrosit alan ve nöronal veya astrositik hücre sayımı) doza bağımlı eğilimleri araştırıldı.

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M., Hirrlinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+metabolism+in+brain+metabolic+interaction+between+astrocytes+and+neurons+in+the+defense+against+reactive+oxygen+species.">Glutathione metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species.</a> Eur J Biochem. 267, 4912-4916 (2000).</li><li>Crist&#242;fol, R. M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

Yazarlar Millipore Corporation&#39;ın tüm çalışanların.

Bugüne kadar astrositler nöronlar ve karışık kültürlerin kullanıldığı nörotoksisite risk değerlendirme çalışması için tasarlanmış, in vitro deneyler sınırlı kalmıştır. Glial hücreleri nöronlar için mekansal ve metabolik destek sağlayan ayrılmaz bir parçasıdır ve nöronal göç, farklılaşma ve sinaptik aktivite de dahil olmak üzere modüle birçok nöronal fonksiyonları, kritik bir rol oynadığı iyi kabul edilir. Glial astrositler birçok toksinler de nöronal doku çevresindeki olumsuz tepkiler inducing yanı sıra teşvik nöronal hoşgörü yeteneğine sahiptir sitokinler ve diğer çözünür faktörlerin de bırakın. Antioksidan savunma bakım ve hücresel enerji düzeyleri gibi astrositik fonksiyonları, değer düşüklüğü görülmüştür ise nöronal-glial etkileşimleri derinliği gösterilmesi, ko-kültür çalışmaları, astrositler nöronlar oksidatif stres toksisite karşı korumak için gösterilen edilmiştir eleştirel nöronal hayatta kalma etkiler. Son çalışmalar, in vitro nörotoksisite test sistemi bir astrositler kullanımı tek başına nöronal hücrelerin daha insan merkezi sinir sisteminin yapısı ve fonksiyonu için daha uygun ispat edebilir ileri sürmüşlerdir. Nöronal astrositik etkileşimleri fizyolojik önemi artan farkındalık rağmen, daha önce sağlam hücreleri bu etkileşimlerin kantitatif analizleri gerçekleştirmek için zor olmuştur. Yüksek İçerik Tarama gelişiyle yeni ve güçlü testlerinin geliştirilmesi bu adrese sağlar. Bu çalışmada, kantitatif analizleri tek bir tahlil içinde birden çok nöronal ve astrositik fenotipleri HCA ile mümkün olduğunu göstermiştir. Primer nöronlar, nöron sayısı, neurite sayısı ve neurite uzunluk olarak nörotoksisite belirteçlerin kantitatif ölçümler gerçekleştirdik. Astrositler, reaktif gliozis nörotoksisite bilinen bir biyomarker, GFAP ifade astrosit numarası ve astrosit morfolojisi değişiklikler ile karakterizedir. Biz bu uç noktalarının her biri kolay HCA ile değerlendirilebilir göstermiştir. Bu çalışmalar, sinir-özgü biyobelirteçlerin HCA nörotoksik uç noktaları kimyasalların hızla çok sayıda ekrana in vitro testler bir pil parçası olarak kullanılan olabilir bu kavramı destekler . Millipore astrositler nöronlar ve ortak kültür HCS222 Yüksek İçerik Analizi kiti yüksek kaliteli algılama reaktifler ve aynı anda nörotoksisite cep telefonu modelleri çok çeşitli βIII-tubulin ve glial fibriller asidik protein (GFAP) profil için protokolleri oluşur. Bu kit ile sağlanan yüksek geçerliliği reaktifler kullanıcı testleri standardize tahlil-tahlil değişkenliği en aza indirmek ve tekrarlanabilir yüksek bir sinyal-zemin oranı ile görüntü oluşturmak için izin verir.

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

Yüksek İçerik Analizi (HCA) testleri tek bir tahlil içinde birden çok hücre fenotipleri ölçümü sağlayan hücreleri ve algılama reaktifleri otomatik görüntüleme ve güçlü görüntü analizi algoritmaları ile birleştirir. Bu çalışmada, nörotoksisite için yeni bir tahlil geliştirmek için HCA kullandı. Nörotoksisite değerlendirme, önemli bir ilaç güvenliği değerlendirmesi bir parçası olarak, çevre koruma çabalarının önemli bir odak olmanın yanı sıra temsil eder. Ayrıca, nörotoksisite de iyi kabul edilir<em&gt; In vitro</emAlzheimer ve Parkinson hastalığı gibi nörodejeneratif hastalıkların gelişimi&gt; belirteç. Son zamanlarda, HCA nöronal tarama uygulaması bildirilmiştir. ΒIII-tubulin nöronal hücrelerin etiketleme, HCA deneyleri nöron sayısı, neurite sayısı ve neurite uzunluğu, nörotoksik etkileri olduğunu göstermemektedir bütün bunlar gibi parametrelerin yüksek verimli olmayan öznel, nicel ölçümleri sağlayabilir. Ancak, astrositler rolü bu modellerin keşfedilmemiş kalır. Astrositler, merkezi sinir sistemi (MSS) homeostazının bakım tamamlayıcı bir rol var ve bunlar toksik maddeler veya hastalık durumlarında tepki aktive nöro ve neurodegradation ile ilişkilidir. GFAP CNS astrositler ağırlıklı olarak ifade bir ara filament proteindir. Astrositik aktivasyonu (gliozis) yaygın astrosit proliferasyon ve hipertrofi eşlik GFAP upregülasyonunun yol açar. Reaktif gliozis Bu işlem, sinir sistemi hasarı erken belirteci olarak öne sürülmüştür. Immunoassay ile GFAP kantitatif için geleneksel yöntem. Bu yaklaşım, hücresel lokalizasyonu, morfolojisi ve hücre sayısı hakkında bilgi sağlamak için bir yetersizlik ile sınırlıdır. Biz HCA bu sınırlamaları aşmak için, ve aynı anda birden çok özellik gliozis ile ilişkili ölçmek için kullanılan olabilir tespit GFAP ifade değişiklikleri, astrosit hipertrofisi ve astrosit proliferasyonu - tek bir tahlil içinde. Ko-kültür çalışmaları, astrositler nöronlar çeşitli toksik hakaret karşı korumak ve eleştirel nöronal hayatta kalma etkisi görülmüştür. Son çalışmalar önerilen olduğunu bir astrositler kullanımı<em&gt; In vitro</em&gt; Nörotoksisite test sistemi sadece nöronal hücrelerin daha insan MSS yapısı ve fonksiyonu için daha uygun ispat edebilir. ΒIII-tubulin ve glial fibriller asidik protein (GFAP) profil Dolayısıyla, biz, bir HCA testi astrositler nöronlar ve ortak kültürü için geliştirilen protokolleri oluşan ve valide, hedef özel algılama reaktifleri. Bu testte, nörotoksisite değerlendirmesi olmayan öznel, yüksek verimli test bir roman temsil nörotoksisite aynı anda analiz, neurite akıbet, gliozis, cep modelleri çok çeşitli nöronal ve astrositik morfoloji ve nöronal ve astrositik gelişim sağlar. Testi, nörolojik araştırma ve ilaç keşfi nörotoksisite ve geliştirilmiş verimlilik gelişmiş algılama için büyük bir potansiyele sahiptir.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Bu makale, bir roman protokol ve reaktif bileşikler ve tedavilerin yüksek içerik analizi kullanarak nöronlar ve astrositler ortak kültürleri üzerinde nörotoksik etkileri hassas ölçümü için tasarlanmıştır ayarlamak açıklamaktadır. Sonuçlar yüksek bir içerik analizi nörotoksisite değerlendirmesi için heyecan verici yeni bir teknoloji temsil ettiğini göstermektedir.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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Research

JoVE Journal

Biology

25.0K Görüntüleme.  Millipore Inc.  日本語 

Video: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Büyüme Konileri Yüksek Çözünürlüklü Görüntüleme için Aplysia Nöronal Hücre Kültürleri

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Biyoloji

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Çözünür Sinyal aracılı Etkileşimleri Araştırılması için Epitel Hücreleri ve Bakteriler mikroakışkan Co-kültür

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Hitachi F-7000 Floresans Spektrofotometre ve PicoGreen Boya kullanarak dsDNA Niceleme

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Toz numuneler mutlak Kuantum Verimi Ölçümü

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

İfade Klonlama tarafından Grb2 Sinyal Kompleksi Eğitim için bir Yüksek içerik Görüntüleme İş Akışı

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Peptid Epitopların Kantitatif Analiz için bir yüksek veri akışı MHC II Bağlanma Deneyi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag Analizi - A Yüksek Verimli, Sc2.0 Genotiplendirme için Real Time PCR Testi

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Zararlı ve Potansiyel Zararlı Bileşenlerinin Toksikolojik etkilerinin değerlendirilmesi için yüksek İçerik Tarama Analizi (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...