Sağlık, NHLBI National Institutes (RD, PY, PS). Amerikan Kalp Derneği (JL). Stanford Üniversitesi, VPUE Grant (JL).

Önceki çalışmada 1 den uyarlanmıştır. 1. SPIO için Annexin V Konjugasyon <ol><li> 2: 0.15 M sodyum periodat (NaIO 4) (ağırlık oranı 04:01 ağırlıkça) ile çözelti içinde karanlıkta 20, 1 saat boyunca SPIO partikülleri (Ocean Nanotech Inc, 5 mg / mL) ° C okside. </li><li> 20 ° C&#39;de 0.15 M sodyum borat (Na 2 B 4 O 7 10H 2 O): ANX protein (ağırlık 1:1 oranı, ağırlık) saflaştırılmış ile 12 saat boyunca okside SPIO inkübe </li><li> Çeker ocak altında sodyum borohidrid (NaBH4) ile 1 saat boyunca daha da azaltmak ve karışım daha sonra 0.15 M Tris-HCl sertleştirme. </li><li> 14.000 xg (75 mikron gözenek Microcon filtresi, Millipore, Billerica, MA) santrifüj ile ANX-SPIO gelen ücretsiz ANX ayırın. </li><li> Daha bir Magnetik Separatör cihazı (Ocean Nanotech, Inc) kullanarak ilişkisiz kirleri kaldırarak bileşik geliştirebilirsiniz. </li><li> Filtre retentat (ANX-SPIO) protein düzeyleri ölçmek-80 azından Bradford protein metodu ve deposu tarafından ° C&#39;de 2 azından% 1 serum albumin ve proteaz inhibitörü (Halt proteaz inhibitör, Termo Scientific, 1-50 mg / mg ANX) ile 0.1 M Tris-HCl içinde mg / ml. </li></ol> 2. Dinamik ışık saçılımı <ol><li> Ayrı 0.1 optik yoğunluğu PBS SPIO ve ANX-SPIO sulandırmak ve Zetasizer Nano DLS makinesi (Malvern Inc, Worcestershire, Birleşik Krallık) 3 dinamik ışık saçılımı analiz yaparlar. </li><li> Z-ortalama parçacık boyutu ve polidispersite indeksi (PDI) ile birlikte, bileşik için monomodal zirveleri (ideal) elde edilir. Tek başına demir oksit Nanopartikülün DLS-ölçülen boyutu bu boyutu karşılaştırın. ANX-SPIO DLS sonuçları homojen bir ANX-SPIO türler 3 yansıtan 0.13 1 bir PDI ile 78 nm konjuge parçacık hidrodinamik çapı gösterdi. </li></ol> 3. Hücre Kültürü, Kültür apoptoz İndüksiyonu ve Doku ve Hücre MRG <ol><li> Kültür yetişkin fare kemik iliği mezenkimal sDMEM içinde TEM hücreleri (MMSC)% 10 FBS ve tercihen pasaj az% 1 penisilin streptomisin (Invitrogen, Inc), 20-30 Nisan ile desteklenmiş. </li><li> DOX (Sigma,% 0.9 tuzlu su içinde 1 mM, ortam içinde 1 uM konsantrasyon) 12 ile 37 ° C &#39;de kat değişen için işleme tabi tutulmasıyla kültürü hücrelerde apoptoza yol. </li><li> ANX-SPIO (aşağıda 3.4 &#39;de açıklanan) ile MR veya TUNEL leke sonra mikroskop kullanılarak (APO-BrdUTM TUNEL Test Kiti, Moleküler Problar / Invitrogen, Eugene, OR) veya ANX-FITC leke sonra birini kullanarak kültürü apoptozisin miktarını ölçmek (Roche, İsviçre). </li><li> 37, 15 dak ° C. için, ANX-SPIO (olarak belirtilmedikçe 10 mg protein / mL, orta), ya da serbest SPIO (5 mg), eşdeğer bir kilo sahip hücrelerin etiketlemek SPIO Nanopartiküllerin difüzyon önlemek için% 1 agaroz 1 mL PBS ile 4 kat ve bindirme yıkayın. Yemekler MRG (aşağıda açıklanmıştır) gerçekleştirin. Alternatif olarak, hücreler yıkayıp DOX ve ANX-SPIO işaretlendikten sonra trypsinize, sonra hücreler izinEppendorf tüplere pelet oluştururlar. </li></ol> 4. Vitro MRG&#39;de <ol><li> In vitro MRG analizi için standart diz alıcı bobini ile bir 1.5 veya 3 Tesla GE Signa EXCITE tarayıcı kullanın. </li><li> Tekrarlama Süresi (TR) 550 ms, Echo Saat (TE) 10 ms, flip açısı 15 °, matris 256 x 256, alan: kültür kaplarına veya tüpleri agar fantom içine ve gradient-eko spoiled gradient dizisi kullanarak görüntüyü yerleştirin of-view 3 cm x 3 cm, kesit kalınlığı 1,3 mm, ortalama sayısı (NEX) 2, aralık 0 5 mm. Görüntü eksenel sabit bir alanı bölgeleri-of-faiz kullanılarak T2 sinyal kaybı, (ROI) (örneğin, 0,8 mm 2) plaka üzerinde hücre katmanı. Agar hayalet gelen kontrol ROI sinyal kullanılarak arka plan için ayarlayın. Fantom çevreleyen havanın sinyal arka plan gürültü ölçün. </li><li> [SI örnek ROI-SI kontrolü ROI] / SI sinyal yoğunluğunu, [arka plan gürültü SI SD] ve: MR Kontrast-Gürültü Oranı (CNR hesaplayınSD) Standart sapma her kültür kaplarına hesaplandı. </li></ol> 5.. Temsilcisi Sonuçlar In vitro T2 * sinyal kaybı ölçüm sinyali geçersiz eser yaratacak homojen bir agar fantom ve hava kabarcıklarının eksikliği gerektirir. Bu eser SPIO hakiki T2 * sinyal kaybı ayırt etmek zordur, ve yorumlanabilir sonuçlar önemlidir. Bir hava eser örnek olarak orijinal demir oksit T2 sinyal kaybı için Şekil 1&#39;de görebilirsiniz. In vitro kültür kaplarına görüntüleme için kalınlığında yaklaşık 1 mm yeterli aksiyel MR dilimleri, yeterince görüntü tüm çanak yapılabilir, böylece her kültürün çanak agar jel uygun bir hacim uygulanır. Şekil 2 beklenen MRG sinyal göstermektedir agar bireysel kültür plakalardan. Int bölgeleri bazı sinyal plakasının kenar varyasyonu, böylece analizi tipik olarak mevcutturerest bu düzensiz alanlardan kaçınmak gerekir. <img alt="Şekil 1" src="/files/ftp_upload/3775/3775fig1.jpg" /> Şekil 1. ANX-SPIO doğan sıçanlardan kültüre kardiyomiyositlerde apoptoz algılar. Şekil 1A kontrol solüsyonu (sol tüp), ANX-SPIO (orta boru), DOX + ANX-SPIO (sağ tüp) ile tedavi edilen pelet yenidoğan kardiyomiyositlerin bir fotoğraf gösteriyor . DOX ve kahverengimsi renk + ANX-SPIO tedavi hücreleri, apoptotik hücrelerde ANX-SPIO bileşiğin artan saklama yansıtan unutmayın. In vitro MR görüntüleri Şekil 1B DOX + ANX-SPIO hücrelerin yoğun T2 sinyal kaybı göstermektedir. Ayrıca, bir hava kabarcığı 4. ve sinyal ANX-SPIO yoksun, ve CNR analizi etkileyebilir benzer 5. Eppendorf tüpleri (küçük siyah sinyal void) arasında görülür. <img alt="Şekil 2" src="/files/ftp_upload/3775/3775fig2.jpg" /> Şekil 2. ANX-SPIObağlama kültürü apoptotik hücreler için spesifik değildir. Şekil 2A ANX-SPIO düşük ve yüksek konsantrasyonlarda (sağ, sol ve sırasıyla) ile ANX-FLUOS boyama veya ANX-SPIO boyama ya maruz kültür kaplarına üzerine apoptotik sıçan neonatal kardiyomiyositler, göstermektedir. Not Farklı T2 floresan ANX (ANX-FLUOS) ve ANX-SPIO yüksek seviyede ve düşük düzeyde tedavi edildi apoptotik hücre * sinyal kaybı (siyah sinyali). Artan T2 sağ * sinyal kaybı daha yüksek ASX-SPIO konsantrasyonları tarafından kapalı yarıştı edilmiş bir belirgin bir azalma oldu ANX-FLUOS sinyali ile ilişkilidir. Şekil 2B ANX-SPIO karşı ANX-FLUOS sinyal ve ayrışma rekabet bağlayıcı göstermektedir sabiti, i K. X-ekseni log ölçeğinde Annexin protein konsantrasyonunu ifade eder. Film 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">ek görmek için buraya tıklayın</a>film.

<ol>
	<li>Dash, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+MRI+probe+to+detect+treatment+of+cardiac+apoptosis+in+vivo.">A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.</a> Magn. Reson. Med. , (2011).</li><li>Khym, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+of+methylamine+with+periodate-oxidized+adenosine+5'-phosphate.">The reaction of methylamine with periodate-oxidized adenosine 5'-phosphate.</a> Biochemistry. 2, 344-350 (1963).</li><li>Liu, Y. Y., Yu, Y., Zhang, G. B., Tang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+controlled+release+of+novel+nanoparticles+based+on+MMA/beta-CD+copolymers.">Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers.</a> Macromol. Biosci. 7, 1250-1257 (2007).</li><li>Seo, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FeCo/graphitic-shell+nanocrystals+as+advanced+magnetic-resonance-imaging+and+near-infrared+agents.">FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents.</a> Nat. Mater. 5, 971-976 (2006).</li><li>Hsiao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+labeling+of+mesenchymal+stem+cells+without+transfection+agent:+cellular+behavior+and+capability+of+detection+with+clinical+1.5+T+magnetic+resonance+at+the+single+cell+level.">Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.</a> Magn. Reson. Med. 58, 717-724 (2007).</li><li>Zhao, M., Beauregard, D. A., Loizou, L., Davletov, B., Brindle, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+apoptosis+using+magnetic+resonance+imaging+and+a+targeted+contrast+agent.">Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.</a> Nat. Med. 7, 1241-1244 (2001).</li><li>van Tilborg, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+annexin+A5-functionalized+iron+oxide+particles+by+apoptotic+Jurkat+cells.">Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells.</a> Contrast Media Mol. Imaging. 4, 24-32 (2009).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+cardiomyocyte+apoptosis+with+a+novel+magneto-optical+nanoparticle.">Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.</a> Magn. Reson. Med. 54, 718-724 (2005).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MRI+detects+low+levels+of+cardiomyocyte+apoptosis+in+a+transgenic+model+of+chronic+heart+failure.">Molecular MRI detects low levels of cardiomyocyte apoptosis in a transgenic model of chronic heart failure.</a> Circ. Cardiovasc. Imaging. 2, 468-475 (2009).</li><li>Dumont, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+death+induced+by+myocardial+ischemia+and+reperfusion:+measurement+with+recombinant+human+annexin-V+in+a+mouse+model.">Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.</a> Circulation. 102, 1564-1568 (2000).</li><li>Blankenberg, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+and+imaging+of+phosphatidylserine+expression+during+programmed+cell+death.">In vivo detection and imaging of phosphatidylserine expression during programmed cell death.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 6349-6354 (1998).</li><li>Schellenberger, E. A., Sosnovik, D., Weissleder, R., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magneto/optical+annexin+V,+a+multimodal+protein.">Magneto/optical annexin V, a multimodal protein.</a> Bioconjug. Chem. 15, 1062-1067 (2004).</li></ol>

Çıkar çatışması ilan etti.

Annexin V SPIO bağlama için tarif konjugasyon yöntemi Annexin yan zincirli amin gruplarının ve SPIO Nanopartikülün karboksil yarımlar yararlanır. Belirli bir oksitleme-indirgeme adımlarında, bu bileşiklerin kovalan bağlantı elde edilebilir, ve elde edilen fonksiyonlandırılmış nanoparçacık izole edilebilir. Bu yöntem, diğer proteinler ve ilgi nanopartiküller üzere genelleştirilmiş olabilir. En önemli aşamaları uygun sıcaklıklarda ve yumuşak karışımı ile ger...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog # </td></tr><tr><td> Süperparamanyetik demir oksit </td><td> Ocean Nanotech, Inc </td><td> ICK-40-005 </td></tr><tr><td> Doksorubisin </td><td> Sigma </td><td> D1515-10mg </td></tr><tr><td> Ayırıcı SuperMag </td><td> Ocean Nanotech, Inc </td><td> N / A </td></tr><tr><td> Zetasizer Nano DLS makinesi </td><td> Malvern, Inc </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

Hücresel apoptoz birçok hastalığın önemli bir özelliktir ve hastalığın klinik belirtileri ortaya çıkmadan bu programlanmış hücre ölümü genellikle oluşur. Onun erken, tersinir aşamalarında apoptoz algılamak için bir araç önleyici veya tedavi edici önlemler kalıcı hasarlardan kalbi korumak için alınabilecek sırasında bir klinik öncesi &#39;pencere&#39; göze olacaktır. Biz burada MR-saptanabilir kontrast ajan olarak hizmet süperparamanyetik demir oksit (SPIO) nanopartiküller için heyecanla apoptoz erken, tersinir aşamalarında hücrelerine bağlanarak insan Annexin V (ANX), birleşmek için basit ve sağlam bir yöntem sunmaktadır. Konjugasyon yöntemi SPIO ve polisakkarit kabuk karboksil grupları okside SPIO nanopartiküller, bir oksidasyon ile başlar. ANX proteini saflaştırılmış ve sonra bir indirgeyici tampon içinde SPIO ile ANX arasında kovalent etkileşimi kolaylaştırmak için bir sodyum borat çözeltisi ortamda eklenir. Sodyum borohydrid ile son bir azalma adıme redüksiyon tamamlamak üzere gerçekleştirilir ve daha sonra reaksiyon söndürüldü. Konjuge olmayan ANX mikrosantrifüj filtrasyon ile karışım kaldırılır. ANX-SPIO ürünün saflığı boyutu ve dinamik ışık saçılması (DLS) ile teyit edilmektedir. Bu yöntem ek olarak, veya modifikasyonu gerektirmez, çapraz bağlı olarak demir oksit partikül konjugasyon yöntemler veya biyotin etiketli nanopartiküller karşıt polisakkarid SPIO kabuk. Sonuç olarak, bu yöntem, ilgi diğer proteinler konjugasyon uzayabilir basit, güçlü bir yaklaşımı temsil eder.

Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Apoptoz Erken teşhis hastalıklarının çeşitli risk altındaki hücre popülasyonu tespit edebilir. Burada bir MR-saptanabilir demir oksit nanoparçacık (SPIO) bir erken apoptozis algılama protein (Annexin V) bağlamak için bir yöntem gösterilmektedir. Bu yöntem MR-saptanabilir moleküler görüntüleme probları üretmek için ilgi diğer proteinlere uzatılabilir.

Bir sentezi<em&gt; In vivo</em&gt; MR-saptanabilir Apoptoz Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

synthesis-of-an-in-vivo-mri-detectable-apoptosis-probe

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Biology

Synthesis of an In vivo MRI-detectable Apoptosis Probe

9.1K Görüntüleme.  Stanford University Medical Center. Apoptoz Erken teşhis hastalıklarının çeşitli risk altındaki hücre popülasyonu tespit edebilir. Burada bir MR-saptanabilir demir oksit nanoparçacık (SPIO) bir erken apoptozis algılama protein (Annexin V) bağlamak için bir yöntem gösterilmektedir. Bu yöntem MR-saptanabilir moleküler görüntüleme probları üretmek için ilgi diğer proteinlere uzatılabilir.

Video: Synthesis of an In vivo MRI-detectable Apoptosis Probe

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Tüm Plazmidler Homemade Sitesi Yönetmen Mutagenez

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biyoloji

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Apoptozis sırasında Mitokondrial Kalsiyum Düzeyleri Dinamik değişiklikler izlenmesi Genetiği Kodlanmış Kalsiyum Sensor Kullanımı

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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 ...

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, ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Aktif Kaspaz 3 Algılama Tüm montaj immünofloresans tarafından Zebra balığı Embriyolar Apaptozun Analizi

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Bir In vivo</emŞu Protein Kompleksler yalıtmak için> Çapraz yaklaşım Drosophila Embriyolar

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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 ...