Name: modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death
Uploaded: 2011-04-24T14:00:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

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.

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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.

<table><tbody><tr><td>1 x PBS-/-</td><td></td><td></td><td></td></tr><tr><td>1 x Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>Annexin V-Alexa Fluor 488</td><td>Molecular Probes, Life Technologies</td><td>A13201</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2% Formaldehyde</td><td>Sigma-Aldrich</td><td>F1268</td><td></td></tr><tr><td>RNase A from bovine pancreas</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td>DR50050</td><td>Dilute 1:60 in 1 x PBS-/-</td></tr></tbody></table>

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 ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

103.1K 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

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Algılama ve yalıtım için yüksek saflıkta apoptotik organlarının

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Biyokimya

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

İzleme bölünmüş Kaspaz-3 Aktivite ve Potasyum kaynaklı Membran depolarizasyon Canlı hücre Görüntüleme ve Cleaveable EPA-boya Substratlar ölümsüzleştirdi oligodendroglial Hücre Apoptoz

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Nörobilim

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

Mitokan Sitotoksisitesinin Doğru Tayini için Otomatik Diferansiyel Nükleer Boyama Denemesi

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Kanser Araştırmaları

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Aktinomisin D ile Tedavi Edilen SiHa Serviks Kanseri Hücrelerinde Apoptotik Biyobelirteçlerin Akış Sitometrik Analizi

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

Bir Hipotalamik Hücre Modeli Apoptosisi Belirleyen Bir Kaspaz Çoğullama Testi Kullanımı

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Böbrek Hücreleri aşırı eksprese eden protein kinaz C izozimlerinin Mitokondriyal Fonksiyonlar ve Hücre Canlılık Değerlendirilmesi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biyoloji

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Kültür hücreleri için Canlılık Tahliller

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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 ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

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İleri Biyoloji

Hücre Biyolojisi

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell&#8217;s membrane, but become &#8220;flipped&#8221; to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured&#8212;a characteristic of both necrosis and late apoptosis.This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

Cell Death Pathways and Annexin V &amp; PI Labeling studies

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...