Bu çalışma Radyasyon Onkolojisi, Alabama-Birmingham Üniversitesi Kapsamlı Kanser Merkezi&#39;nde, Dövüş Çocuk Kanser Vakfı ve Gabrielle&#39;in Angel Vakfı (ESY için.) Bölümü IMPACT Ödülü ile desteklenmiştir.

1. Hücre Kültürü <ol><li> Kültür nöronal hücrelerin ve onları gerektiği gibi davranın. </li><li> 15 ml&#39;lik tüp içine hasat hücreleri: aspiratı ortam, fosfat ile durulama (PBS, kalsiyum ve magnezyum serbest) tamponlu tuzlu su, 15 ml tüp içinde toplamak ve uygun bir serum ihtiva eden ortam ile nötralize tripsin, tripsin ekleyin. </li><li> 5 dakika boyunca 1000 xg Spin. </li><li> Medya aspire. </li><li> PBS içinde hücreler yeniden süspanse edin. </li><li> 5 dakika boyunca 1000 xg Spin. </li><li> Taze PBS içinde medya ve tekrar süspansiyon hücreleri aspire. </li><li> Bir hemasitometre veya tercih ettiğiniz hücre sayıcı kullanarak hücreleri saymak. </li><li> Hücre numuneleri deney başlamadan hemen önce hazırlanmalıdır. </li><li> Tüm numuneler, morötesi ışık DNA hasarı önlemek için ya da koyu sarı ışık altında ele alınmalıdır. </li></ol> 2. Comet Assay 1. Slayt Hazırlama <ol><li> % 1 agaroz (1X Tris Base, Borik asit, EDTA, TB ve 1 g / 100 ml eritinE) 3 dakika süreyle mikrodalga tüm granül kayboluncaya kadar. </li><li> Dip erimiş agaroz içine slaytlar ve bir tarafı silin bir Kimwipe ile temizleyin. Şeffaf bir film kuruması için agaroz izin verin. Bu muhafaza edilebilir ve önceden kızak içinde yapılabilir. </li></ol> 2. Nötr Comet Assay <ol><li> Kullanımdan önce en az 1 saat boyunca 4 ° C sıcaklıkta erimesi Chill çözeltisi (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris baz,% 1 sodyum lauril sarkosinat, ve% 1 Triton X-100, pH 10). </li><li> 2.2.2. Tüm granül kayboluncaya kadar bir mikrodalga% 1 düşük erime noktası agaroz (1X Tris Bankası&#39;ndaki 1 g / 100ml, Borik asit, EDTA) 3 dakika eritin. Agaroz kuyruklu kuyruk yapay indüksiyon önlemek için ° C&#39;de bir su banyosu içinde 37 kadar soğutuldu gerekmektedir. İdeal olarak, agaroz kullanımdan önce yarım saat için soğutulması gerekmektedir. </li><li> 2.2.3. , Ml başına 100,000 hücre olup, böylece hücre süspansiyonu ile seyreltilir. Düşük erime noktası agaroz (37 ° C) ile hücre süspansiyonu Kombine1:10 (v / v), girdap kısaca, oranı ve hemen Comet slayt üzerine 50 ul pipetle. Örnek alan üzerinde eşit hücre süspansiyonu yaymak için pipet ucu tarafını kullanın. Her tedavi grubu en az üç kopya halinde olmalıdır. </li><li> Bir daire slayt periferinde görünene kadar Sıra 30 dakika boyunca buzdolabında düz kayar. </li><li> Prechill 4 karanlıkta 30 dakika için bu çözelti içinde lizis çözelti daldırın ve slaytlar ° C&#39;de (ya da bir soğutma cihazı olarak). </li><li> Kapalı dökün veya lizis çözüm aspirat ve 1X nötr elektroforez tamponu (Tris baz, Borik asit, EDTA, 1X TBE) ekleyin. Buzdolabında yarım saat bu tampona slaytlar bırakın. </li><li> Elektroforez odasında önceden soğutulmuş 1X Nötr Elektroforez Tamponu (TBE), elektroforez slayt tepsisine yerleştirin slayt ekleyin. Onlar elektrotlar eşit uzaklıkta olacak şekilde slaytlar hizalayın. </li><li> 1X nötr elektroforez slaytlar üzerinde 0,2 inç kadar tampon dökün. Aşırı tampon arası olacakelektroforezi ile Fere. </li><li> Cm başına 1 V (elektrot elektrot ölçülür) güç kaynağı gerilimini ayarlayın ve karanlıkta 4 ° C (veya soğuk oda) 30 dakika boyunca çalıştırın. </li></ol> 3. Alkali Comet Assay <ol><li> Kullanımdan önce en az 1 saat boyunca 4 ° C sıcaklıkta erimesi Chill çözeltisi (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris baz,% 1 sodyum lauril sarkosinat, ve% 1 Triton X-100, pH 10). </li><li> Tüm granül kayboluncaya kadar 3 dakika boyunca mikrodalga içinde% 1 düşük erime noktası agaroz (1X Tris Bankası&#39;ndaki 1 g / 100 ml, Borik asit, EDTA) eritin. Sonra en az 30 dakika süreyle 37 ° C su banyosunda soğutulur. </li><li> 1:10 (v / v), girdap kısaca, bir oranında erimiş düşük bir erime noktasına sahip agaroz 1 x 105 / ml arasındadır (37 ° C) hücreleri ile birleştirin ve hemen Comet slayt üzerine 50 ul pipetle. Örnek alan üzerinde eşit hücre süspansiyonu yaymak için pipet ucu tarafını kullanın. Her tedavi grubu en az üç kopya halinde olmalıdır. </li><li> Sıra kaydırdıes düz 30 dakika buzdolabında bir daire slayt periferinde görünene kadar. </li><li> Lizis çözelti içinde önceden soğutulmuş ve 4 ° C&#39;de bırakmak karanlıkta gece boyunca, 1 saat için Immerse kayar. </li><li> Lizis çözüm slaytları çıkarın, slaytlar boşaltın ve alkali gevşemek adım öncesinde kalan deterjan ve tuzları kaldırmak için 5 dakika süreyle soğuk nötralizasyon tamponu ile bir kez yıkayın. </li><li> Bir jel elektroforez odasında Sıra slaytlar slaytları yukarıda 0.5 cm geçmeyecek şekilde önceden soğutulmuş taze yapılmış elektroforez tamponu (300 mM NaOH, 1 mM EDTA,&gt; 13 pH) ile doldurulur. Onlar elektrotlar eşit uzaklıkta olacak şekilde slaytlar hizalayın. </li><li> Slaytlar DNA ve alkali sorumlu hasar ifade çözülmesi için izin vermek için karanlıkta 30 dakika süreyle alkalin tampon bekletin. </li><li> Cm başına 1 V (elektrot elektrot ölçülen) güç kaynağı gerilimi ayarlayın ve 4 az 30 dakika çalıştırın ° C (veya soğuk oda). </li></ol> 4. Tespit ve Staining Hücreler <ol><li> Aşırı Elektroforez Tampon boşaltın </li><li> Daldırın oda sıcaklığında 5 dakika boyunca önceden soğutulmuş damıtılmış su içinde kayar. </li><li> Oda sıcaklığında 5 dakika boyunca önceden soğutulmuş% 70 etanol içinde Immerse kayar. </li><li> Gecede Kuru örnekleri. Parlak ışık slaytlar maruz bırakmayın. Numuneler bu aşamada puanlama için oda sıcaklığında ay boyunca muhafaza edilebilir. </li><li> Buzdolabında 20 dakika SYBR Green (PBS içinde seyreltilmesi 1X) daldırarak slaytlar Leke. </li><li> Slaytları çıkarın ve onları karanlıkta tamamen kurumasını bekleyin. Tamamen kuru agaroz şeffaf hale gelecektir. </li></ol> 4. Görüntü Toplama ve Analizi <ol><li> Yeşil filtre (Zeiss AxioVision) ve Comet Assay yazılım (Algısal Instruments) kullanılarak analiz ayarlanır floresan mikroskop kullanılarak görüntü elde edin. </li><li> &quot;Kuyruklu kafa&quot; (çekirdeği) üzerine tıklayın ve yazılımı içinde ortalama kuyruk momenti ve DNA miktarı dahil parametreleri hesaplarçekirdek. </li><li> Tedavi başına en az 200 hücre analiz edin. </li><li> Microsoft Excel&#39;e veri verme. </li><li> Her tedavi grubu ve uygun arsa verileri için kuyruk momenti ortalama hesaplayın. </li></ol> 5. Temsilcisi Sonuçlar Nöron hücreleri üzerinde kuyruklu tahlil analizinin bir örneği Şekil 2 ve Şekil 3 &#39;de gösterilmiştir. Bu durumda, nöronal hücrelerin ışınlama DNA hasarına neden olmaktadır. Hücreler elektrik alanına maruz gibi, DNA daha sonra Comet Test yazılımı kullanılarak analiz edilmiştir boyut farklılıklara bağlı olarak farklı hızlarda geçirir. Daha fazla DNA hasarı, uzak DNA çekirdek dışında geçirir. Bu sonradan yüksek kuyruk anda yazılım ve sonuçları tarafından alınır çekirdeğinde floresan yoğunluğu azalır. Tablo 1 comet assay analizi ve Şekil 4 bir temsilci tablo gösteriyor bir temsilcisi gösterirniteliksel grafiği comet assay ile ölçülen radyasyon sonrasında nöronal hücrelerde DNA tahribatına karşılaştırarak. <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Şekil 1"/> Şekil 1. Kuyruklu testin Akış şeması. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Şekil 2,"/> Şekil 2. Nöron hücrelerinin Örnek görüntü (A) olmaksızın ve (B) kuyrukluyıldız kuyruk ile. <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Şekil 3"/> Comet Assay yazılımı kullanarak Şekil 3. Comet assay analizi. (A) görüntü Temsilcisi ekran görüntüleri Carl Zeiss floresan mikroskop kullanılarak kazanılmış ve Comet kullanılarak analizTest yazılımı. (B) nukleus ya da &quot;kuyruklu kafa&quot; (yeşil daire) tıkladığınızda bir floresan harita ve grafik (sarı daire içinde) ve (C) veri tablosu (kırmızı daire) oluşturur. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Şekil 4,"/> Şekil 4. Nötr comet assay kullanarak ışınlanmış ve ışınlanmamış nöronal hücrelerde DNA tahribatına çözümlenmesiyle elde edilen ortalama kuyruk momenti komplo ile elde Temsilcisi grafiği. Beklendiği gibi ışınlanmış nöronal hücrelerde yüksek ortalama kuyruk momenti tarafından gösterildiği gibi, 3 Gy radyasyon (x-ray), DNA hasarı indükler. , ** P &lt;0.01 - ortalama kuyruk momenti (Standart hata + /) olduğu gösterilir. <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Tablo 1"/> Tablo 1. </strong&gt; ışınlanmış ve ışınlanmamış nöronal hücreler üzerinde kuyruklu testi analizi ile elde Örnek tablo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50049/50049table1large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> .

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</ol>

Çıkar çatışması ilan etti.

Comet assay bireysel hücrelerin analiz eşsiz kapasitesine sahiptir. Bu sitotoksik ajanlara diferansiyel tepki göstermek hücrelerinin alt popülasyonlarının belirlenmesi avantajlıdır. Birkaç pratik sınırlamaları dikkate alınmalıdır. Ayrı ayrı değerlendirilebilir hücrelerin sayısı her bir bağlı olarak değişebilir. Bir popülasyon içindeki DNA hasarı varyans varsa örneklem büyüklüğü artırılmalıdır. Nekrotik ya apoptotik hücrelerin baskın varlığı hata katacak yana Yaşayabilir tek hücre süspansiyonu Bu tahlil için kritik öneme sahiptir. Lizis ve elektroforez adımları apoptotik hücreler, küçük DNA parçacıkları (50kb daha küçük) ve mitokondriyal DNA kurtulur. Böylece, comet assay 1,3-11 tarafından algılanmaz. Yukarıda da belirtildiği gibi, comet assay alkali labil siteleri ve ilgili DNA-DNA/DNA-protein çapraz bağlantılar dahil tek ve çift zincir kırıkları, tespit etmenin en etkin yoludurDNA. Nötr comet assay çift iplikli sonları sadece algılama sağlayan ise, alkali tahlil tek iplikçik tatili yanı sıra çift iplikli sonları algılama sağlar: Burada iki ayrı protokol ana hatlarıyla. Enzim modifiye comet assay oksidatif DNA adducts tespit uygulandı yapabilirsiniz. Örneğin, Endo III (endonükleaz) ve hOGG1 (glikozilaz) ile muamele glikol timin ve urasil glikol 12,13 ve 8-okso-7 ,8-dihydroguanine (8-oxoGua) 14,15 içeren pirimidin okside tanımak. Formamidopyrimidine DNA-glikozilaz (APG) benzer bir şekilde kullanılabilir. Bu değiştirilmiş test için, enzim ile muamele adımı öncesinde çözülmesi. Lezyona spesifik enzimlerinin ilave kuyruklu test de 13 duyarlılığını arttırır. Burada DNA hasarının tanımlayıcısı olarak kuyruk momenti kullanın. Kuyruk an aşağıdaki formül kullanılarak hesaplanır: kuyruk DNA yüzdesi len çarpılırkuyrukluyıldız kuyruk 1,3-7 arasında gth. Kuyrukluyıldızın kuyruğu (kuyruk uzunluğu) uzunluğu DNA göç uzak noktaya çekirdeğin merkezinden mesafedir. Baş uzunluğu çekirdeğin çapı. Kafa yoğunluğu ve kuyruk yoğunluğu kuyruklu yıldızın baş ve kuyruk ortalama piksel yoğunluğu, sırasıyla. Toplam alan kuyruklu 1,3-7 toplam yüzey alanıdır. Böyle γ-H2AX odakları boyama ve pulsed-field jel elektroforez gibi DNA hasarlarının ölçme diğer yöntemler olmasına rağmen, bu ölçümlerin yanı 4,6,11,16,17 kendi dezavantajları var. Örneğin, sık sık replikasyon stres γ-H2AX seviyeleri 18 ölçü içinde bir karıştırıcı faktör olduğunu. Γ-H2AX odaklar oluşumu DNA tamir proteinleri işe bağlı olduğu için, DNA tamir proteinleri ile bir kondanse kromatin yapısı toplamak için başarısızlık de yanlış düşük γ-H2AX odaklar seviyelerine sebep olabilir. Benzer şekilde, pulsed-field jel electrophoresis çok zaman yoğundur ve büyük DNA moleküllerinin 19,20 ayrılması için sadece faydalıdır. Böylece, comet assay DNA hasarı ve onarımı 10,11,21 nispeten kolay, verimli ve ucuz, doğrudan ve duyarlı bir ölçüsüdür.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> 1.5 ml&#39;lik tüplere </td><td> Santa Cruz Biotechnology, Inc </td><td> Sc-200271 </td><td></td></tr><tr><td> 10X TBE (Tris baz, borik asit, EDTA) </td><td> Fisher Scientific </td><td> BP13331 </td><td></td></tr><tr><td> 15 ml tüp </td><td> Fisher Scientific </td><td> 0553851 </td><td></td></tr><tr><td> Agaroz </td><td> Sigma </td><td> A5093 </td><td></td></tr><tr><td> Alüminyum folyo </td><td> Fisher Scientific </td><td> 01213101 </td><td></td></tr><tr><td> Deney şişesi </td><td> Fisher Scientific </td><td> FB102300 </td><td></td></tr><tr><td> Santrifüj </td><td> Thermo Scientific </td><td> 75004261PR </td><td></td></tr><tr><td> Comet Assay yazılımı </td><td> Comet Assay IV Görüntü Analiz Sistemi </td><td></td><td></td></tr><tr><td> Silindir </td><td> Fisher Scientific </td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td> Elektroforez odası </td><td> Thermo Scientific </td><td> 09528101 </td><td></td></tr><tr><td> Etanol </td><td> Fisher Scientific </td><td> A407P4 </td><td></td></tr><tr><td> Floresan mikroskop </td><td> Zeiss Axio Vizyon </td><td></td><td> Yeşil filtre ile herhangi bir floresan mikroskop yeterli olacaktır </td></tr><tr><td> Hemasitometre </td><td> Fisher Scientific </td><td> 0267152 </td><td></td></tr><tr><td> Düşük erime noktası agaroz </td><td> Promega </td><td> V2111 </td><td></td></tr><tr><td> Mikrodalga </td><td> Sears </td><td></td><td></td></tr><tr><td> Fosfat ücretsiz tuzlu, kalsiyum içermeyen, magnezyum tamponlu </td><td> HyClone </td><td> SH3025601 </td><td></td></tr><tr><td> Güç kaynağı </td><td> BioRad </td><td> 1645050 </td><td></td></tr><tr><td> Buzdolabı </td><td> Sears </td><td></td><td></td></tr><tr><td> Cetvel </td><td> Staples </td><td></td><td></td></tr><tr><td> Slayt </td><td> Fisher Scientific </td><td> 12550143 </td><td></td></tr><tr><td> Sodyum klorür </td><td> Sigma </td><td> S7653 </td><td></td></tr><tr><td> Sodyum hidroksit </td><td> Sigma </td><td> S5881 </td><td></td></tr><tr><td> Sodyum lauril sarkosinat </td><td> Fisher Scientific </td><td> S529 </td><td></td></tr><tr><td> SYBR Green </td><td> Invitrogen </td><td> S7585 </td><td></td></tr><tr><td> Tepsi </td><td> FisOnu Bilimsel </td><td> 15242B </td><td></td></tr><tr><td> Tris Baz </td><td> Sigma </td><td> T6066 </td><td></td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> T8787 </td><td></td></tr><tr><td> Tripsin </td><td> HyClone </td><td> SH3023601 </td><td></td></tr><tr><td> Girdap </td><td> Fisher Scientific </td><td> 02216108 </td><td></td></tr><tr><td> Su banyosu </td><td> Fisher Scientific </td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

Ilaçların bir dizi DNA onarım yolakları hedef ve hücre DNA hasarı oluşturarak öldürmeye teşvik. Böylece, doğrudan DNA hasarı ölçmek için süreçlerin kapsamlı değerlendirilmiştir. Geleneksel yöntemler zaman alıcı, pahalı, kaynak yoğun ve kopyalayan hücrelerin gerektirir. Buna karşılık, kuyruklu testi, tek hücre jel elektroforezi testi, DNA hasarı ve onarımı daha hızlı, non-invaziv, ucuz, direkt ve duyarlı bir ölçüsüdür. DNA hasarı yanı sıra DNA tamir bütün formları bu güçlü tekniği kullanarak tek hücre düzeyinde görüntülenebilir. Comet assay yatan prensip DNA hasarı bu organizasyon bozan ise intakt DNA çok sipariş olmasıdır. Hasarlı DNA&#39;nın agaroz matriks içine girer ve bir elektrik alanına maruz kaldığında, negatif yüklü DNA pozitif yüklüdür katoda doğru göç eder. Büyük hasarsız DNA ipliklerini çok çekirdek göç etmek mümkün değildir. DNA hasaruzak bozulmamış DNA daha seyahatlerinizde küçük DNA parçaları oluşturur. Comet Assay, bir görüntü analiz yazılımı, tedbirler ve çekirdeğin dışına göç etmiş DNA ile DNA&#39;dan genel floresan yoğunluğu karşılaştırır. Göç DNA&#39;dan Floresan sinyal DNA hasarı ile doğru orantılıdır. Uzun parlak DNA kuyruk artmış DNA hasarı anlamına gelir. Ölçülen parametreler bazıları kuyruk DNA&#39;sının miktarı ve kuyruk, kuyruk uzunluğundaki DNA dağıtımı ve DNA yüzdesi her ikisi de bir ölçüsüdür kuyruk an vardır. Bu testte DNA hasarının çözünürlüğü tamir yerini almıştır anlamına beri yanı DNA onarım ölçmek için sağlar. Duyarlılık sınırı diploid memeli hücre 1,2 başına yaklaşık 50 iplikli kırılır. Örneğin etoposid gibi herhangi bir DNA hasarına neden olan ajan ile muamele Hücreler, bir pozitif kontrol olarak da kullanılabilir. Böylece comet assay DNA hasarı ölçmek için hızlı ve etkili bir yöntemdir.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Comet assay alkali labil siteleri ve hipokampal nöronlar da dahil olmak üzere tüm hücrelerde DNA üzerinde DNA-DNA/DNA-protein çapraz bağlantılar dahil tek ve çift zincir kırıkları, tespit etmenin en etkin yoludur. Yöntem DNA hasar miktarının farklılıklara bağlı olarak, bir elektrik alanı içinde DNA diferansiyel göç yararlanır.

Comet Assay kullanma Hipokampal Nöronlar DNA Hasarı assaying

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Nanyang Technological University

Research

JoVE Journal

Neuroscience

27.3K Görüntüleme.  University of Alabama-Birmingham. Comet assay alkali labil siteleri ve hipokampal nöronlar da dahil olmak üzere tüm hücrelerde DNA üzerinde DNA-DNA/DNA-protein çapraz bağlantılar dahil tek ve çift zincir kırıkları, tespit etmenin en etkin yoludur. Yöntem DNA hasar miktarının farklılıklara bağlı olarak, bir elektrik alanı içinde DNA diferansiyel göç yararlanır.

Video: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Nörobilim

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

Vitro DNA hasarı kuyruklu yıldız tahlil kullanarak değerlendirmek

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Kanser Araştırmaları

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Nöromodülasyon ve Mitokondrial Ulaştırma: Uzun Süreleri içinde Hipokampal Nöronlar Canlı Görüntüleme

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: İnsan Microarrayed hücrelerde DNA hasarına ölçülmesi için bir Yüksek verim 96 Oyuklu Platformu

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Biyomühendislik

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Prenatal Fareler gelen Hipokampal Nöronlar izolasyonu ve Kültürü

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

DNA Hasarının Değerlendirilmesinde Kuyruklu Yıldız Testinde Donmuş Dokunun Kullanımı

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Genetik

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Sistemik Oksidatif Stresin bir Dolaylı Tedbir olarak Comet Assay

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Biyoloji

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Sıçan Karaciğer DNA Strand Breaks ve Oksidatif DNA Hasar Ölçüm Vivo Alkali Comet Assay ve Enzim-modifiye Alkali Comet Tahlili

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Hücresel DNA Hasarını Değerlendirmek için Yüksek Verimli Bir Kuyruklu Yıldız Testi Yaklaşımı

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Hücrelerde DNA Hasarı Tespiti için Comet Deneyi

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...