Bu araştırma NIH, ncı ve CCR Intramural araştırma programı tarafından desteklenmiştir. Tüm yazarlar NIH, ncı ve CCR Intramural araştırma bursu aldı.

1. hazırlamak reaktifler <ol><li>1 x PBS <ol><li>seyreltik 100 mL 10 x PBS 900 mL dH 2 O ile ve pH 7.4 pH metre kullanarak için ayarlayın. Mağaza oda sıcaklığında.</li>
	</ol></li> <li>Lizis çözüm (LS) <ol><li>2.5 M NaCl, 100 mM disodyum EDTA, 10 mM Tris Bankası ve 900 mL dH 2 O 200 mM NaOH hazırlamak; genellikle tamamen erimesi karışım izin verin için yaklaşık 20 dakika sürer. PH pH metre kullanarak 10 için ayarlayın. % 1 sodyum loril sarcosinate ve % 1 eklemek Triton X-100 ve son ses seviyesini 1000 ml. 4 ° c için kullanmadan önce en az 30 dk serin.</li>
	</ol></li> <li>Alkali Elektroforez çözüm (AES), pH &#62; 13 <ol><li>200 mM NaOH ve 1 mM disodyum EDTA 800 mL dH 2 O. ayarlama pH hazırlamak ve pH olduğundan emin olun &#62; 13. 1000 ml nihai ses düzeyini ayarlayın. Kullanmadan önce taze olun ve serin 4 ° c kullanmadan önce en az 30 dk için.</li>
	</ol></li> <li>Tarafsız Elektroforez sollution (NES) <ol><li>hazırlamak 1000 mL tarafsız Elektroforez arabellek 100 mM Tris Bankası ve 300 mM sodyum asetat 1000 mL dH 2 O. ayarlama 9.0 için pH buzul Asetik asit ile karıştırma tarafından. 4 ° c için kullanmadan önce en az 30 dk serin.</li>
	</ol></li> <li>DNA yağış çözüm (DPS) <ol><li>10 mL 7,5 M amonyum asetat stok hazırlanması. 50 mL için DNA yağış çözeltisi, 6.7 mL 7,5 M amonyum asetat 43.3 mL % 95 etanol ile karıştırın. Mağaza oda sıcaklığında.</li>
	</ol></li> <li>Staining çözüm <ol><li>ekleyin 1 µL 10.000 yeşil flüoresan nükleik asit leke (örneğin, SYBR yeşil) 30 mL Tris-EDTA arabellek (10 mM Tris-HCl, 1 mM disodyum EDTA, pH 7,4) ve store x 4 ° C. koru dan ışık.</li>
	</ol></li> <li>Özel %1 düşük erime <ol><li>% 1 düşük erime noktası özel (100 mL dH 2 O 1 g) bir mikrodalga eritebilir. Özel özel tamamen erimiş olduğundan emin olmak için her 15-20 s girdap. 37 ° C su banyosu için en az 20 dk kullanmadan önce özel yerleştirin.</li>
	</ol></li> <li>Önceden sıcak pipet ipuçları <ol><li>P200 pipet ipuçları dar uçları 3 mm ve 37 ° c sıcak özel pipetting önce kekemelik.</li>
	</ol></li></ol> 2. Kuyruklu yıldız slaytlar hazırlayın <ol><li><ol><li>Melt % 1'özel (100 mL dH 2 O 1 g) 2-3 dk ya da özel tamamen erimiş olana bir mikrodalga kaplama kaydırın. Cam mikroskop slaytlar özel içine daldırma ve hav bırakmayan mendil kullanarak slayt bir tarafı yok.</li>
		<li>Slaytlar kuruması veya daha hızlı kurutma için 50 ° c ısı için düz bir yüzey üzerinde yatıyordu; şeffaf özel film kurutma sonra oluşmuş olmalıdır. Kaplamalı slaytlar 37 ° C kullanmadan önce yerleştirin.</li>
	</ol></li> <li>Tek hücre süspansiyonlar hazırlanması <ol><li>kültür ve tedavi gliyom hücre <ol><li>10 ile desteklenmiş DMEM-Ham F-12 orta U251 MG hücrelerde kültür % FBS, 100 U/mL penisilin ve 10 µg/mL streptomisin, 37 ve # 176; C % 5 CO 2.</li>
			<li>1 mL tripsin 3 dakikadır kullanarak hücreleri sindirmek ve tripsin FBS ile DMEM-Ham F-12 orta kullanarak etkisiz hale getirin. Toplamak 300 x g 4 dk de 15 mL tüp, spin, orta Aspire edin ve 2 x 10 5 hücre/mL 1 x PBS hücreleri askıya alma. 
			Not: Hücre örnek hemen tahlil başlamadan önce hazırlanmalı ve tüm örneklerini ortamda ışık DNA hasarları önlemek için bir koyu veya soluk işleneceğini.</li>
			<li>1:10 (v/v) oranında (37 ° C'de) %1 erimiş düşük erime noktası özel hücre süspansiyon birleştirin, karışımı yavaşça yukarı ve aşağı pipetting ve hemen bir slayda 30 µL pipet. Tarafını kullanıyorsunuz pipet ucu ince bir tabaka oluşumu sağlamak için özel/hücre karışımı spread.</li>
			<li>10 dk. 30 dk jelleşme zaman artan yüksek nem içeren ortamlardan örneklerin yapışma geliştirir düz 4 ° C'de karanlık slayt yer.</li>
			<li>4 ° c LS içinde belgili tanımlık karanlık bir gecede 1 h için slayt bırakın.</li>
		</ol></li></ol></li></ol> 3. Tek hücre Elektroforez <ol><li>devam et alkalin (Adım 3.2) veya nötr (Adım 3.3) kuyruklu yıldız tahlil</li> <li>alkali kuyruklu yıldız tahlil için <ol><li>yavaşça slaytlar LS kaynağından çıkarın, aşırı arabellek drenaj ve yavaşça AES içinde 4'te 1 h için bırakın ° C DNA gevşemek izin vermek için. Slaytları karanlıkta bırakın.</li>
		<li>Ekle önceden soğutulmuş AES, Elektroforez slayt tepsisine (Bu Elektroforez birim boyutuna bağlıdır) slaytlar yukarıda 0.5 cm fazla, slaytlar içinde ve bir kap ile kapak yerleştirin. 1 V/cm (uzunluk elektrot arasındaki) güç Besleme gerilimi ayarlamak ve çalıştırmak için 30 dk 4'te ° C.</li>
		<li>Drenaj aşırı Elektroforez çözüm slayttan. DH 2 O oda sıcaklığında her 5 min için iki kez slaytları yavaşça bırakın.</li>
		<li>% 70 etanol oda sıcaklığında 5 min için slaytları yavaşça bırakın. Adım 4'e devam.</li>
	</ol></li> <li>Tarafsız kuyruklu yıldız tahlil için <ol><li>yavaşça slaytları LS kaynağından çıkarın, aşırı arabellek drenaj ve yavaşça NES içinde 4'te 30 dk için bırakın ° C. Keep slayt karanlıkta.</li>
		<li>Ekle önceden soğutulmuş tarafsız Elektroforez arabellek Elektroforez slayt tepsisinde (Bu Elektroforez birim boyutuna bağlıdır) slaytlar yukarıda 0.5 cm fazla, slaytlar içinde ve bir kap ile kapak yerleştirin. 1 V/cm (uzunluk elektrot arasındaki) güç Besleme gerilimi ayarlamak ve çalıştırmak için 45 dk 4'te ° C.</li>
		<li>Drenaj slaytları aşırı arabelleğinden. Yavaşça DPS slaytları, oda sıcaklığında 30 dakika bırakın.</li>
		<li>% 70 etanol, oda sıcaklığında 30 dk için slaytları yavaşça bırakın. Adım 4'e devam.</li>
	</ol></li></ol> 4. Kuyruklu yıldız slaytlar leke <ol><li>kuru slaytlar için 10-15 dk içinde belgili tanımlık karanlık 37 ° C'de.</li>
	<li>Yer 50-100 µL yeşil flüoresan nükleik asit çözüm her boyama kurutulmuş özel ve 15 dakika içinde belgili tanımlık karanlık oda sıcaklığında leke.</li>
	<li>Slaytları kısaca dH 2 içinde O durulayın ve tamamen 37 ° C'de karanlık kuru. Resim alma ve analiz için devam.</li>
</ol> 5. Görüntü toplama ve analizi Not: görselleştirme ve miktar olan DNA epifluorescence mikroskobu ve kuyruklu yıldız tahlil yazılım dayalı (Tablo malzemeleri görmek) 12 .
<ol><li>Yer slaytları bir slayt tutucu mikroskopla üstünde. Özel jel objektif lens dönük olduğundan emin olun. Rastgele bir floresan mikroskop ile 10 x objektif lens kullanarak lekeli kuyruklu yıldız slaytlardan çekim. Kenarları ve herhangi bir hava kabarcıkları çevresinde kaçının.</li>
	<li>Emin olun her kuyruklu yıldız kuyruk yatay olarak dağıtılır. Kuyruklu yıldız kafaları köken sol ve sağ kuyruk.</li>
	<li>Her resim parlak DNA leke ve koyu arka plan ile ikili bir TIF biçiminde kaydedin. Yüklemek için yazılım kullanarak görüntüleri &#34; seçin dosyaları analiz etmek için &#34; araç çubuğunun solunda bulunan düğme. ( şekil 1) bir görüntü görünüm pencere belirmelidir.</li>
	<li>Ölçüm çerçevesi ekrana çizdiğiniz ve boyutuna uygun olarak hücre kuyruklu yıldız ayarlayın. ' I tıklatın &#34; ayarla &#34; baş, kuyruklu yıldız ve kuyruk görüntü göre eşik ayarlamak için düğme, o zaman tıkırtı &#34; başlatmak ölçümleri &#34; düğmesini ( şekil 1).</li>
	<li>Çerçeve kullanarak bir hücre seçin ve fare ile tıklayarak ölçüm harekete geçirmek &#34; kuyrukluyıldız tahlil &#34; düğme; bir yoğunluk görüntü üzerinde gösterir &#34; profiller &#34; penceresi seçili ölçüm parametreleri ile. Sonuçlar'ı tıklatarak kaydedilebilmesi için &#34; sonucu depolamak &#34; düğmesini ( şekil 1). 
	Not: Yazılım kuyruklu yıldız kuyruk, kuyruklu DNA, kuyruk moment (TM) ve zeytin kuyruk an (OTM) yüzdesi dahil parametreleri hesaplar. Kuyruk anlar formüller tarafından takip hesaplanır: <img alt="Equation 1" src="/files/ftp_upload/56450/56450eq1.jpg" /> <img alt="Equation 2" src="/files/ftp_upload/56450/56450eq2.jpg" /></li> <li>en az 50 hücreleri analiz tedavi başına.</li>
</ol>

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., Collins, A. 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S., 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=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. 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Yazarlar ifşa gerek yok.

Kuyruklu yıldız tahlil tek ve iki iplikçikli DNA sonları hücresel düzeyde ölçmek için etkili bir araçtır. Tahlil yaygın olarak13genotoxicity ve biomonitoring ile ilgili olarak, temel lezyonlar, DNA Glossar, ilaç geliştirme ve alkali hassas siteler arasında değişen çalışmalarda "altın standart" olarak uygulanmıştır. Bu da çalışmanın, sırasıyla alkali ve tarafsız kuyruklu yıldız deneyleri, iki ayrı adım adım protokollerde gösterdik. Tek hücre Elektroforez, flore...

Kuyruklu yıldız tahlil ilk Ostling ve Johanson tarafından 1984 yılında çekirdeği bir nötr koşul1altında üzerinden göç DNA parçaları göstererek geliştirilmiştir. Teknik bir alkali durumu önemli ölçüde artan özgüllük ve tekrarlanabilirlik tahlil2gösterilen Singh vdtarafından daha sonra geliştirildi. O zamandan beri tarafsız kuyruklu yıldız tahlil çoğunlukla ise alkali kuyruklu yıldız tahlil DNA hasar, tek gibi küçük miktarlar için daha duyarlı ve Çift Kişilik iplikçik DNA tatili, alkali değişken siteler, DNA-DNA çift iplikçikli DNA tatili, algılamak için kullanılır veya DNA-protein cross-linking ve DNA tek iplikçikli kırılmalar eksik eksizyon ile ilişkili siteleri3,4onarın. Her iki deneyleri görselleştirme parçalanmış DNA'ın izin ve kantitatif DNA hasarı değerlendirmek için basit bir yol sağlar. Kuyruklu yıldız tahlil içinde in vitro ve in vivo genetik toksikolojik çalışmalar için hassas bir yöntemi olarak kabul edilir ve erken uyuşturucu-aday seçimi, çevresel izleme, insan biomonitoring gibi farklı araştırma alanları için geçerlidir, ve araştırma DNA hasarı ve5onarım temel.
Tahlil bir elektrik alanı altında parçalanmış DNA (olarak da bilinen "kuyruklu yıldız kafa") çevrili vücut dışına geçirir ve özel jel (olarak da bilinen "kuyruklu yıldız kuyruğu") bir DNA leke oluşturur ilkedir. Nükleotit boyama ile DNA hasar ölçüde "kuyruklu yıldızlar bu tek hücre Elektroforez tarafından kurulan" analiz ederek sayısal. Kuyruk an hesaplanması daha fazla DNA hasar farklı deneysel grupları arasında karşılaştırmak için yardımcı olabilir. DNA hasar tespiti geleneksel yöntemlere göre kuyruklu yıldız tahlil doğrudan, hassas, ucuz ve oldukça basit olduğunu.
Radyoterapi ve chemotherapies kanser tedavisi için ortak stratejiler tek iplikçik oluşturarak ve Çift Kişilik iplikçik DNA kromozom6' tatili. DNA onarım inhibitörleri son gelişme kombinasyon kemoterapi tarafından daha etkili bir genotoksik etkiyi sağlar ve bu nedenle, potansiyel olarak anemi, enfeksiyon ve kemik iliği baskılanması7, gibi sistemik yan etkileri azaltır 8. bu çalışmada, bir Poli (ADP-riboz) polimeraz (PARP) inhibitörü, olaparib (Ola)9incelenmesi gösterdik. PARP bol bir nükleer protein ve Poli (ADP-riboz) polimer10oluşturarak DNA baz eksizyon tamiri için sorumludur. Temozolomide (TMZ) sözlü olarak kullanılabilir bir bölücü ve yaygın olarak tümörü hasta tedavi için kullanılan. Kuyruklu yıldız tahlil DNA hasarı ölçmek için kullanarak, biz olaparib derinden temozolomide ile birleştirerek olaparib/temozolomide kombinasyon tedavisi tümörü, tedavi etmek için etkili bir strateji olduğunu göstermektedir DNA hasarı gliyom hücrelerdeki geliştirir göstermek temozolomide yalnız11ile karşılaştırıldığında.

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">10x PBS(Ca++, Mg++ free)</td>
			<td style="width:101px;">TEKnova</td>
			<td style="width:119px;">P0196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:101px;">TEKnova</td>
			<td style="width:119px;">E0308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">72068</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium lauryl sarcosinate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">L7414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">93443</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">32318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glacial acetic acid</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">695092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium acetate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A1542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR Green</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:119px;">S33102</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Low melting point agarose</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:119px;">16520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:119px;">16500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">95% ethanol</td>
			<td style="width:101px;">WARNER-GRAHAM</td>
			<td style="width:119px;">#64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:101px;">GIBICO</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Consumables</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glass tissue slides</td>
			<td style="width:101px;">ELECTRON MICROSCOPY SCIENCES</td>
			<td style="width:119px;">63422-11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kimwipes</td>
			<td style="width:101px;">KIMberly-Clark</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL Microcentrifuge Tubes</td>
			<td style="width:101px;">DENVILLE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette Tips</td>
			<td style="width:101px;">SHARP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipments</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microwave</td>
			<td style="width:101px;">Avanti</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Waterbath</td>
			<td style="width:101px;">PRECISION</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horizontal electrophoresis chamber</td>
			<td style="width:101px;">TREVIGEN</td>
			<td style="width:119px;">Cometassay ES II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power supply</td>
			<td style="width:101px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:101px;">Quincy Lab</td>
			<td style="width:119px;">Model 12-140E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent microscope</td>
			<td style="width:101px;">Zeiss</td>
			<td style="width:119px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipettor</td>
			<td style="width:101px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating in vitro dna damage using comet assay

DNA hasar her hücre için ortak bir fenomen onun ömrü ve genomik DNA'ın kimyasal yapısı bir değişiklik tanımlanır. Gibi radyo ve kemoterapi, kanser tedavileri kanser ilerleme sınırlamak için hücre döngüsü tutuklama ve Apoptozis yol ek DNA hasar çok büyük miktarda tanıtmak. DNA hasarı kantitatif değerlendirilmesi deneysel kanser tedavisi sırasında genotoksik aracısı etkinliğini haklı göstermek için önemli bir adımdır. Bu çalışmada, biz bir tek hücre elektroforez yöntemi, olarak da bilinen tek ölçmek kuyruklu yıldız tahlil odaklanmak ve iki iplikçikli DNA vitrotatili. Kuyruklu yıldız tahlil verimli ve gerçekleştirmek kolay bir DNA hasarı miktar yöntemidir ve düşük saat/bütçe talepleri ve yüksek tekrarlanabilirlik vardır. Burada, olaparib/temozolomide kombinasyon tedavisi U251 tümörü hücrelere genotoksik etkisini değerlendirerek kuyruklu yıldız tahlil preklinik bir çalışma için yardımcı programı vurgulayın.

Kuyruklu yıldız tahlil yürütme ve veri analizi (şekil 1) için adım adım iş akışı mevcut protokolünü açıklar. Alkali ve tarafsız kuyruklu yıldız deneyleri sonuçları doksorubisin tedavi U251 hücreler (1 µM, 20 h) kuyrukluyıldızın kuyruğunun daha uzun ve daha yüksek DNA şiddeti, parçalanmış DNA kemoterapi (Şekil 2) nedeniyle önemli bir birikimi düşündüren vardı gösterdi.
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Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

Vitro DNA hasarı kuyruklu yıldız tahlil kullanarak değerlendirmek

Kuyruklu yıldız tahlil tek ve çift iplikçikli DNA içeren DNA hasarı tatili algılamak için verimli bir yöntemdir. Kemoterapi tedavi edici etkisini değerlendirmek için kanser hücrelerinin DNA hasarı ölçmek için alkali ve tarafsız kuyruklu yıldız deneyleri tarif.

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 ...

evaluating-in-vitro-dna-damage-using-comet-assay

Research

JoVE Journal

Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

36.9K Görüntüleme.  National Cancer Institute. Kuyruklu yıldız tahlil tek ve çift iplikçikli DNA içeren DNA hasarı tatili algılamak için verimli bir yöntemdir. Kemoterapi tedavi edici etkisini değerlendirmek için kanser hücrelerinin DNA hasarı ölçmek için alkali ve tarafsız kuyruklu yıldız deneyleri tarif.

Video: Evaluating In Vitro DNA Damage Using Comet Assay

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Yukarı doğru hareket: Bir basit ve esnek Vitro üç boyutlu işgali tahlil iletişim kuralı

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Kanser Araştırmaları

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

Sözde Hemangioblastoma tahlil çimlenme küresel kullanarak neovaskülarizasyon Vitro performansını değerlendirmek için kapsamlı bir yordam

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Kanserle ilgili yorgunluk mitokondriyal işlev rolünün değerlendirilmesi

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Gerçek zamanlı hafiye-in Vitro tümör hücre Apoptosis içinde CD8 tarafından indüklenen+ T hücreleri Myeloid hücrelerin tümör infiltre bağışıklık baskılayıcı işlevlerini çalışmaya

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In vitro assay tümör çalışma-macrophage etkileşim

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

Organoid Kültürü Kullanarak Fare Prostat Epitel Hücrelerinin Ayırt Etme Kapasitesinin Değerlendirilmesi

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Yapısal DNA Alterasyon Analizi ve Hasta Kaynaklı Ksenogreftler Kullanılarak Kanserde Hedefli Tedavilerin Test Edilmesi

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

Kolorektal Kanser Hücrelerinin Üç Boyutlu Küresel Kültürlerinde Probiyotiklerin Hücresiz Süpernatantı Kullanılarak Hücre Ölümünün Değerlendirilmesi

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

İnsan Kolon Kanseri Hücrelerinde DNA Hasarı ve Onarım Foci İmmünofloresan Görüntüleme

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.