Bu çalışmada, Doğa Bilimleri ve Mühendislik Konseyi, Kanada (NSERC) araştırma bursu ve bir Alberta Tarım DRB Finansman Konsorsiyum hibe tarafından desteklenmiştir. AMR bir NSERC Vanier Kanada Yüksek Lisans Bursu, Alberta asistanlığa bir üniversite ve bir Kraliçe Elizabeth II yüksek lisans bursu ile desteklenmektedir.

Bu prosedür, 500 mcL silikonlu tüpler veya 6 ml polistiren yuvarlak alt FACS tüpler yapılabilmektedir. İkincisi, diğer prosedürleri ile birlikte canlılığını analizi yaparken normal olarak kullanılır. 6 ml polistren yuvarlak alt FACS tüpler için verilen tüm birimleri. 500 mcL silikonlu borular için tüm birimler, 1 / 5 oranında azaltır. Flow sitometri analizi için optimum konsantrasyonu 200 mcL hacim başına 2-4 x 10 6 hücre. Hücre kaybı bu yordamı neden olabilir ve böylece biz her numune prosedürün başlangıcında 4 x 10 6 hücre oluşur. 1. Hücre Hazırlık <ol><li> Hasat hücreleri - karşılık gelen hücre hatları veya birincil hücre izolasyonların için belirli prosedürleri bakın. </li><li> 10 dakika boyunca 335 x g örnekleri santrifüj ve süpernatantı süzün. </li><li> Süspanse edin hücreleri 2 mL 1 x fosfat tamponlu salin (PBS) / - (kalsiyum, magnezyum). </li><li> 10 dakika boyunca 335 x g örnekleri santrifüj ve süpernatantı süzün. </li><li> 1 ml 1 x Annexin V bağlayıcı tampon hücrelerin tekrar </li><li> 10 dakika boyunca 335 x g örnekleri santrifüj ve süpernatantı süzün. </li><li> 100 mcL 1 x Annexin V bağlayıcı tampon içinde süspanse edin hücreleri. </li></ol> 2. Annexin V / PI Uygulama Leke <ol><li> Üreticinin tavsiyelerine göre Annexin V [örneğin 5 mcL Annexin V Alexa Fluor 488 (Molecular Probes, A13201)]. </li><li> Karanlık tüpler 15 dakika oda sıcaklığında inkübe edin. </li><li> Her reaksiyon tüpüne 1 x Annexin V bağlayıcı tampon 100 mcL ekleyin. Her bir tüp yaklaşık 200 mcL olmamalıdır. </li><li> 1 x Annexin V bağlayıcı tamponu (yani x Annexin V bağlayıcı tampon 9 mcL 1 ile 1 mcL PI) 01:10 seyreltilmiş PI 4 mcL (Sigma, Kedi # P-4864-10ML) ekleyin. Bu, her örnek 2 mcg / ml son bir PI konsantrasyon verecektir. </li><li> Karanlık tüpler 15 dakika oda sıcaklığında inkübe edin. </li><li> Hücreleri yıkamak için 500 mcL 1 x Annexin V bağlayıcı tampon ekleyin. </li><li> 10 dakika boyunca 335 x g örnekleri santrifüj ve süpernatantı süzün. </li><li> 500 mcL 1 x Annexin V bağlayıcı tampon ve% 1 formaldehit (fiksatif) çözümü oluşturmak için 500 mcL% 2&#39;lik formaldehit içinde süspanse edin hücreleri. Tüpler nazik hafifçe vurarak karıştırın. </li><li> 10 dakika boyunca buz üzerinde örnekleri sabitleyin. Alternatif olarak, numuneler 4 gecede saklanan ° C karanlıkta. İkinci yöntem seçilirse, Annexin V / PI (tazminat kontrolleri dahil olmak üzere) etiketli tüm tüpler tutarlı emin olun. </li><li> Her bir örnek ve hafifçe vurarak yavaşça karıştırın - / - 1 ml 1 x PBS ekleyin. </li><li> Sekiz dakika için 425 x g tüpler Santrifüj ve süpernatant süzün. </li><li> 2.10 ve 2.11 adımları tekrarlayın. </li><li> Tüp Flicking tarafından süspanse edin pelet. </li><li> 50 mcg / mL son bir konsantrasyon elde etmek 1:100 sulandırılmış RNaz A (Sigma, R4642) 16 mcL ekleyin. 37 az 15 dakika boyunca inkübe ° C. </li><li> / - 1 ml 1 x PBS ekleyin ve hafifçe vurarak yavaşça karıştırın. </li><li> Sekiz dakika için 425 x g tüpler santrifüjleyin. </li><li> Numuneler analiz edilecek hazır. Annexin / PI boyama diğer prosedürleri ile paralel olarak yapılıyorsa Alternatif olarak, numuneler sonraki boyama adımlar için kullanılan olabilir. </li></ol> 3. Temsilcisi Sonuçlar: Hücre tipleri ve hücre hatları, yanlış pozitif PI boyanma değişen derecelerde örnekleri Şekil 1&#39;de gösterilmiştir. (Şekil 2B) RNaz tedavi uğramadı hücrelere göre yanlış pozitif olayların sayısında önemli bir azalma yanlış pozitif olaylar için hesap için, hücreler fikse edildi ve daha sonra bu sonuçlar RNaz A. ile tedavi. Şekil 2C hiçbir RNaz tedavisi görmüş hücrelere kıyasla RNaz tedavi uygulandı hücreleri temsil eden görüntüler gösterir. Şekil 3 fiksasyon ve RNaz tedavi adımları ile modifiye Annexin V / PI protokolü, yanlış pozitif boyanma olayların sayısının sınırlandırılması geleneksel protokoller üzerinde nasıl geliştirdiğini gösteriyor. <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="Şekil 1" /> Şekil 1. Konvansiyonel propidium iyodür boyama protokolleri önemli sayıda yanlış pozitif olayların yol açar. Daha önce, hayvan modellerinin çok geniş bir yelpazeden yanı sıra çeşitli hücre hatları 10 birincil hücreler boyunca yanlış pozitif boyanma yaygınlığı değerlendirilir . Temsilcisi görüntüleri üç benzersiz hücre popülasyonları (RAW makrofaj ve iki birincil hücreler fare kemik iliği makrofajlar (BMM) ve Japon balığı primer böbrek makrofajlar (PKM) yaygın olarak kullanılan hücre hattı) yanlış pozitif boyanma ölçüde göstermektedir. Gerçek pozitif olayların nükleer bölmesi içinde beklenen PI ve DRAQ5 arasındaki ortak lokalizasyonu (PI ve DRAQ5 sarı alanlar arasında ko tasvir) gösterir. DRAQ5 yüksek membrane geçirgen boya, canlı ve ölü hücreleri 10,11,12 hem doğru lekeleri nükleer bölmesi. Ko DRAQ5 daha kolay görsel tespiti için leke yeşil pseudocolour verildi. <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="Şekil 2" /> Şekil 2. PI nükleer boyanma doğruluğunu artırma. (A) DRAQ5 için iyi karakterize nükleer lekeleri çok sayıda (BrdU, 7-AAD ve DAPI) benzerlik derecesine göre PI nükleer boyanma doğruluğu değerlendirildi. BrdU hızla çoğalan hücrelerin DNA içine dahil olup, sadece nükleer bölmesi gibi içinde leke sentetik bir nükleozid. 7-AAD, sağlam bir plazma zarı geçemez, PI benzer bir DNA yüksek afinite ve vardır. DAPI DNA için de güçlü bir ilgisi vardır ve en yaygın olarak kullanılan nükleer floresan mikroskopi leke. Doğru RAW 264,7 makrofaj hücre çekirdeğinde leke yeteneğini vurgulayarak, BrdU, 7-AAD, DAPI ve DRAQ5 arasında benzerlik derecesi&gt;% 99 oldu. Buna karşılık, geleneksel protokoller dayalı sitoplazmik PI boyama DRAQ5 için göreli boyama yüzde benzerlik belirgin bir azalmaya yol açar. Fare kemik iliği makrofajlar (BMM) ve Japon balığı primer böbrek makrofajlar (PKM): (B) Benzer sonuçlar test iki birincil hücreler gözlenir. 50 mcg / ml RNaz A boyama prosedürü içinde belirli bir aşamada dahil edilmesi, nükleer olmayan PI boyama kaldırır ve DRAQ5 boyama göre benzerlik derecesi önemli ölçüde artırır. (C) primer böbrek makrofajlar Temsilcisi görüntüleri ve RNaz tedavi olmadan hücrelerin ölçüde benzerlik göstermektedir. Tedaviler arasında farklılıklar daha kolay görsel tespiti için, DRAQ5 yeşil verildi. Sarı alanlar arasında ko BrdU ve DRAQ5 ve PI ve DRAQ5 betimliyor. <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="Şekil 3" /> Şekil 3. Modifiye Annexin V / PI yanlış apoptozis / nekrotik olaylar önemli ölçüde azaltır. İlköğretim goldfish böbrek makrofajlar (PKM), geleneksel ve modifiye Annexin V / PI protokolleri ile boyandı. Saçılım Annexin V / PI goldfish PKM leke betimliyor. Soldaki saçılım PKM hücrelerinin geleneksel Annexin V / PI boyama (RNaz yok) gösterir. Sağda saçılım modifiye Annexin V / PI protokolü (RNaz) ile boyandı PKM hücreleri gösterir. Ayrıca yanlış pozitif lekeleri kaldırılması nedeniyle PI boyama belirgin bir azalma RNaz sonuçları. PKM hücreler daha sonra farklı kültürlerin erken atalarıdır içinde nüfus (AP), monositler (Mono) ve olgun makrofajlar (Mat mac) dayalı analiz edildi. Yanlış pozitif olayların kaldırılması nedeniyle, olumlu PI olayların sayısında azalma, küçük erken progenitör hücreleri daha büyük olgun makrofaj hücreleri daha belirgindir. Geleneksel protokollerine dayalı sonuçlar hatalı hücre ölüme daha olgun makrofaj altkümelerini için pozitif bir korelasyon önerilen olurdu.

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. 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Annexin V / PI protokolü burada sunulan geleneksel protokollerin değiştirilmiş bir sürümü ve PI için yüksek afinite sitoplazma RNA varlığı dikkate alır. Nükleer PI boyama boyama işlemi geç% 1 formaldehit fiksasyonu adım aşağıdaki RNaz A (50 mcg / ml) Giriş hassasiyeti önemli ölçüde artırır. Nükleer PI boyama veya plazma zarı Annexin V boyama olumsuz etkileri görülmektedir. RNaz bir tedaviye gerek kalmadan, PI leke sonuçlarının% 40 kadar downstream sonuçlar 10 bir potansiyel olarak anlamlı ve negatif bir etkiye yol açan yanlış pozitif olaylar, yol açabilir. Modifiye Annexin V / PI boyama protokol basit ve hücre tipleri (İlköğretim hücreleri geniş bir yelpazede etkili bir şekilde kullanılır olmuştur: fare kemik iliği makrofaj ve dalak, domuz akciğer ikamet hücreleri, akciğer alveolar makrofajlar, mezenterik lenf nodu izolatları, periferik kan lökosit splenositlerin; tavuk blastodermin hücreler; goldfish birincil böbrek makrofajlar; sazan periferik kan lökosit, Hücre hatları: RAW 264,7 makrofajlar, Jurkat T hücreleri, domuz 3D4/31 makrofajlar, CCL-71 Japon balığı yüzgeci fibroblastlar, 3B11 kedibalığı B hücreleri 10). Hücreler farklı birincil hücreler genellikle yanlış pozitif olaylar (10) daha fazla sayıda olan yalancı pozitif PI boyama, etkilenen dikkat etmek önemlidir. Bu hücresel popülasyonlar arasındaki farklılıklar, yanlış pozitif PI boyama kısmen hücre boyutunda farklılıklar olabilir, ancak RNA içerik farklılıkları da neden olabilir. Bu yordamı, birleşme deneysel sistemler diğerleri arasında kullanmak, genotoksik stres geçiren hücreler, hücre döngüsü tutuklanması gibi timidin ya da hidroksiüre gibi ilaçlar, viral-enfekte olmuş hücreleri ve gelişim ilerlemesi embriyonik hücreler üzerinde çalışmaları ile tedavi edilen hücreler için özellikle ilgili olarak hücresel RNA sentezi ayrık değişiklikler ile karakterizedir.

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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

Hücresel apoptozis Çalışmaları flow sitometri tabanlı yöntemler sunulmasından bu yana önemli ölçüde etkilendi. Propidium iyodür (PI) hücreler, plazma zarı bütünlüğü ve geçirgenliği 1,2 farklılıklar üzerinden, canlı, apoptotik veya nekrotik eğer belirlemek için yaygın Annexin V ile birlikte kullanılır. Annexin V / PI protokolü apoptotik hücre 3 çalışmak için yaygın olarak kullanılan bir yaklaşımdır. Hücreleri 4,5 yaşayan boya hariç kapasitesini dayalı, ekonomik, istikrarlı ve hücre canlılığı iyi bir göstergedir, çünkü PI diğer nükleer lekeleri göre daha sık kullanılır. Bir hücreye girmek için PI yeteneği membran geçirgenliği bağlıdır; PI, sağlam bir plazma zarı 1,2,6 varlığı nedeniyle canlı ya da erken apoptotik hücre leke yapmaz . Geç apoptotik ve nekrotik hücreler, plazma ve nükleer membran bütünlüğünü sağlayarak, PI, membranlar yoluyla geçmesi, nükleik asitler içine enterkale ve kırmızı floresan 1,2,9 gösterilecek 7,8 azalır . Ne yazık ki, bu geleneksel Annexin V / PI protokollerini 10 sitoplazmik bölmesi içinde RNA PI boyama ile ilgili önemli sayıda yanlış pozitif olaylar (% 40), yol bulabilirsiniz. Gösteren yüksek oluşumu 10: (sitoplazmik oranları &lt;0.5 nükleer) Birincil hücre ve hayvan modellerinde geniş bir yelpazede büyük hücreler ile hücre hatları etkilenir. Burada, geleneksel yöntemlere kıyasla hücre ölümü değerlendirilmesi için önemli bir gelişme sağlayan bir değiştirilmiş Annexin V / PI yöntemi göstermektedir. Bu protokol RNaz, boyama aşağıdaki hücre içine girişini teşvik etmek için hücre sabitleme sırasında hücresel geçirgenlik değişikliklerin yararlanır. Zamanlama ve RNaz konsantrasyonu her ikisi de bir sitoplazmik RNA kaldırılması için optimize edilmiştir. Sonuç olarak geleneksel Annexin V / PI protokolleri (sitoplazmik PI boyama &lt;% 5 olaylar) üzerinde önemli bir gelişmedir.

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Hücre ölümü değerlendirilmesi için doğru bir yöntem açıklanmıştır. Protokol birincil hücre hücre hatları ve geniş bir yelpazede hayvan modellerinde% 40 yalancı pozitif olaylar kadar ekran geleneksel Annexin V / propidium iyodür (PI) protokolleri, üzerine geliştirir.

Hücre Ölümü doğru değerlendirilmesi için modifiye Annexin V / Propidium İyodür Apoptoz Testi

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

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Research

JoVE Journal

Biology

102.4K Görüntüleme.  University of Alberta. Hücre ölümü değerlendirilmesi için doğru bir yöntem açıklanmıştır. Protokol birincil hücre hücre hatları ve geniş bir yelpazede hayvan modellerinde% 40 yalancı pozitif olaylar kadar ekran geleneksel Annexin V / propidium iyodür (PI) protokolleri, üzerine geliştirir.

Video: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Kardiyojenik İndüksiyon Verimlilik miktarının için modifiye Fare Embriyonik Kök Hücre tabanlı Testi

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biyoloji

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

A β-glucuronidase (GUS) Tabanlı Hücre Ölümü Testi

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Sitokrom Sitoplazmik Mikroenjeksiyon Apoptoz aktivasyonu C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Embriyolar yılında Programlı Hücre Ölümü Algılama LysoTracker kullanımı ve Embriyonik Kök Hücreler Farklılaşan

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Mozaik Yetişkin Canlı Görüntüleme Domates / GFP-FLP / FRT Yöntemi<em&gt; Drosophila</em&gt; Fotoreseptör Hücreler

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Takip inişi, Apoptosisi tersine Cep Survival Fenomeni için Stratejiler

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Bir iyodür-Sarı floresan Protein-Gap Junction hücreler arası iletişim tahlil

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Yetişkin Kas Kök Hücre İşlevselliğinin Değerlendirilmesi için Tek Miyofiber Kültür Tahlil Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

CLON-G ile Nötrofil Yaşam Süresinin Uzatılması ve In Vitro Spontan Ölüm Testi

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...