Name: mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay
Uploaded: 2018-05-01T12:30:00.000+00:00
Duration: 5 min 53 s
Description: Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Bu çalışmada Shandong seçkin bilim adamı (JQ201421) ve NSFC (81371226) hibe verilmesi gelen hibe tarafından desteklenmiştir.

1. mitokondri yalıtım
Not: Tüm çözümleri ve ekipman olmalı 0 - 4 ° C olarak precooled ve buz tuttu.
<ol><li>10 cm hücre kültür çanak başına tohum yaklaşık 3 x 106 SH-SY5Y hücreleri. Yüksek glikoz Dulbecco hücrelerde Kültür'ın kartal orta % 10 fetal sığır serum, penisilin (100 U/mL) ile modifiye-streptomisin (100 µg/mL). Gecede %5 CO2 içeren bir kuluçka 37 ° C'de korumak. Not: en az 1-2 x 107 SH-SY5Y hücreleri her yalıtım tahlil için ihtiyaç vardır.</li>
	<li>Orta kaldırmak ve yaklaşık 1 mL 10 cm hücre kültür tabak içinde buz soğuk PBS hücrelerle 3 kez yıkayın.</li>
	<li>Buz soğuk PBS yeni 1 mL 10 cm hücre kültür çanak içine ekleyin ve yeni bir 1,5 mL tüp içine yapışık hücreleri kazımak. 4 ° C'de 10 dakika 800 x g, santrifüj</li>
	<li>Santrifüjü sırasında proteaz inhibitörleri hisse senedi 50 μL ile takıma 5 mL mitokondrial yalıtım arabellek (250 mM Sükroz, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazin ethanesulfonic asit), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA) hazırlamak çözüm (100 x konsantrasyon; 1 EDTA ücretsiz tablet GKD2O 500 µL içinde çözünmüş).</li>
	<li>Süpernatant kaldırmak ve Pelet mitokondrial yalıtım arabelleği 1 mL ile resuspend. Buz 30 dk için 1,5 mL tüp kuluçkaya.</li>
	<li>Bir cam homogenizer (15-25 vuruş) askıya alınan hücrelerle yukarı ve aşağı el ile buzda parçalayıcı. Not: hava kabarcığı yok hücreleri homojen olduğundan emin olun. Sağlam hücreleri ve hücre çekirdeği bared tarafından mikroskop görsel olarak saymak ve bunu teyit > % 90 hücre kırılma oluştu.</li>
	<li>Homogenate 1.5 mL tüp ve 4 ° C'de (çekirdek ve kalan sağlam hücreleri) enkaz kaldırmak için 5 min için 1000 x g, santrifüj içine aktarın.</li>
	<li>Yeni 1,5 mL tüp ve 5 min için 1000 x g, santrifüj içine süpernatant transfer 4 ° C.</li>
	<li>Yeni tüp ve 15dk için 14.000 x g, santrifüj süpernatant transfer 4 ° C.</li>
	<li>Süpernatant kaldırmak ve mitokondri 1 mL mitokondrial yalıtım arabelleği ile içeren Pelet resuspend.</li>
	<li>Çözüm 14.000 x g 4 ° C de 15 dakika santrifüj kapasitesi mitokondri çökelti.</li>
	<li>Elde edilen mitokondri Pelet buz tuttu ve 50-100 μL KCl medya (125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 20 mM HEPES, 5 mM Glutamat, 5 mM malat ve 2 mikron rotenon, pH 7,4) herhangi bir eşek önce Pelet hacmine göre resuspended ay.</li>
	<li>Mitokondrial çözüm 5 μL standart BCA tahlil OD562 ölçme tarafından mitokondrial protein konsantrasyonu ölçmek için kullanın bir plaka okuyucu13kullanarak.</li>
</ol>2. mitokondrial Ca2 + saklama kapasite tayini
<ol><li>KCl ortam hazırlamak ve Ca2 +Ekle-0.5 mikron deneme önce son bir konsantrasyon yeşil flüoresan boya bağlama.</li>
	<li>1 mL izole mitokondri (0.4 mg/mL) 0.5 mikron Ca2 +içeren KCl medya aktarma-her 6-şey tabak içine yeşil flüoresan boya de bağlama.</li>
	<li>Mitokondri ve Ca2 +kuluçkaya-yeşil flüoresan boya oda sıcaklığında 1 dk. eklemek 4 μL aliquots 20 mm CaCl2 çözüm (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2 için ortam ışığı korunuyorsunuz 6-şey plaka bağlama 20 mM HEPES, 5 mM Glutamat, 5 mM malat, pH 7,4) her 6-şey plakasına iyi otomatik kullanarak dağıtmak 200 nmol Ca2 +mitokondriyal /mg protein tanıtmak için ayarlama.</li>
	<li>Floresans Spektrometre Floresans değişiklikleri izlemek için her 3 kullanın s 506 bir uyarma dalga boyu ile 2 min için nm ve emisyon dalga boyu 531 nm. Plaka için 3 600 RPM sallayarak ile donatılmış s yazılım (şekil 1) tarafından kontrol edilen kaynaklar arasında.</li>
	<li>Her şey için bir ek 4 μL aliquot 20 mM CaCl2 çözüm ekleyin ve her 3 Floresans değişiklikleri izlemek s 2 dakika süreyle Floresans zamanla artar kadar bu adımı yineleyin. Not: Mitokondri ile artan kayıt Floresans tarafından belirtilen ek Ca2 + zorlanmaktadır. MPTP açıldığında, Floresans zamanla artar.</li>
	<li>Toplam miktarı mPTP Ca2 + saklama kapasitesi açana kadar eklendi Ca2 + yorumlamak.</li>
</ol>3. tespiti Ca 2 +-mitokondrial şişme indüklenen
<ol><li>Deneme önce KCl ortam hazırlamak.<ol>
<li>İzole mitokondri (0.4 mg/mL) 1 mL KCl medyada de her 6-şey plaka aktarın.</li>
	</ol>
</li>
	<li>20 mm CaCl2 sulu çözüm (500 nmol/mg mitokondrial protein) 10 μL mitokondrial şişme tetiklemek için mitokondrial protein ekleyin.</li>
	<li>540 Absorbans azalma kaydetmek nm her 3 s 10 dk (Şekil 2) için yazılım tarafından kontrol Mikroplaka okuyucu. Floresans değişiklikleri arasında mitokondri iç zar solutes bir akını tarafından kaynaklanır. Not: 540 azalmış Absorbans nm karşılık gelen şişmiş mitokondri. Eğer okuyucu bir düşüş Absorbans gözlemleme, bir daha uzun aralığı okuma veya okuma zamanı kabul edilebilir.</li>
</ol>

<ol>
	<li>Rossi, C. S., Lehninger, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+respiratory+stimulation,+accumulation+of+Ca+++and+phosphate,+and+oxidative+phosphorylation+in+rat+liver+mitochondria.">Stoichiometry of respiratory stimulation, accumulation of Ca++ and phosphate, and oxidative phosphorylation in rat liver mitochondria.</a> J Biol Chem. 239 (11), 3971-3980 (1964).</li><li>Gunter, T. E., Buntinas, L., Sparagna, G., Eliseev, R., Gunter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+transport:+mechanisms+and+functions.">Mitochondrial calcium transport: mechanisms and functions.</a> Cell Calcium. 28 (5-6), 285-296 (2000).</li><li>Crofts, A. R., Chappell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Ion+Accumulation+and+Volume+Changes+of+Isolated+Liver+Mitochondria.+Reversal+of+Calcium+Ion-Induced+Swelling.">Calcium Ion Accumulation and Volume Changes of Isolated Liver Mitochondria. Reversal of Calcium Ion-Induced Swelling.</a> Biochem J. 95, 387-392 (1965).</li><li>Wojda, U., Salinska, E., Kuznicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+ions+in+neuronal+degeneration.">Calcium ions in neuronal degeneration.</a> IUBMB Life. 60 (9), 575-590 (2008).</li><li>Celsi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+calcium+and+cell+death:+a+deadly+triad+in+neurodegeneration.">Mitochondria, calcium and cell death: a deadly triad in neurodegeneration.</a> Biochim Biophys Acta. 1787 (5), 335-344 (2009).</li><li>Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., Fiskum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+potentiates+the+maximal+calcium+uptake+capacity+of+neural+cell+mitochondria.">Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.</a> Proc Natl Acad Sci U S A. 93 (18), 9893-9898 (1996).</li><li>Rajdev, S., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+green-5N,+a+novel+fluorescent+probe+for+monitoring+high+intracellular+free+Ca2++concentrations+associated+with+glutamate+excitotoxicity+in+cultured+rat+brain+neurons.">Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons.</a> Neurosci Lett. 162 (1-2), 149-152 (1993).</li><li>Stout, A. K., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+calcium+indicators+underestimate+increases+in+intracellular+calcium+concentrations+associated+with+excitotoxic+glutamate+stimulations.">High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations.</a> Neuroscience. 89 (1), 91-100 (1999).</li><li>Bambrick, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A+increases+mitochondrial+calcium+uptake+capacity+in+cortical+astrocytes+but+not+cerebellar+granule+neurons.">Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.</a> J Bioenerg Biomembr. 38 (1), 43-47 (2006).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-permeable+peptide+antioxidants+targeted+to+inner+mitochondrial+membrane+inhibit+mitochondrial+swelling,+oxidative+cell+death,+and+reperfusion+injury.">Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury.</a> J Biol Chem. 279 (33), 34682-34690 (2004).</li><li>Muriel, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+free+calcium+levels+(Rhod-2+fluorescence)+and+ultrastructural+alterations+in+neuronally+differentiated+PC12+cells+during+ceramide-dependent+cell+death.">Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.</a> J Comp Neurol. 426 (2), 297-315 (2000).</li><li>Allman, R., Hann, A. C., Phillips, A. P., Martin, K. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Azotobacter+vinelandii+with+correlation+of+Coulter+cell+size,+flow+cytometric+parameters,+and+ultrastructure.">Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.</a> Cytometry. 11 (7), 822-831 (1990).</li><li>Wiechelman, K. J., Braun, R. D., Fitzpatrick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+bicinchoninic+acid+protein+assay:+identification+of+the+groups+responsible+for+color+formation.">Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation.</a> Anal Biochem. 175 (1), 231-237 (1988).</li><li>Petronilli, V., Nicolli, A., Costantini, P., Colonna, R., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+permeability+transition+pore,+a+voltage-dependent+mitochondrial+channel+inhibited+by+cyclosporin+A.">Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.</a> Biochim Biophys Acta. 1187 (2), 255-259 (1994).</li><li>Pastorino, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+consequences+of+the+sustained+or+transient+activation+by+Bax+of+the+mitochondrial+permeability+transition+pore.">Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.</a> J Biol Chem. 274 (44), 31734-31739 (1999).</li><li>Gunter, T. E., Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+possible+functions+of+mitochondrial+Ca(2+)+transport+mechanisms.">Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.</a> Biochim Biophys Acta. 1787 (11), 1291-1308 (2009).</li></ol>

X. güneş deneyler denetlenir. Wei Li deneyler yapılır. Chen Zhang ve X. güneş kağıt yazdı.

Burada, mitokondrial Ca2 + saklama kapasitesi tahlil ve Ca2 +için basit ve etkili iletişim kuralı açıklanan-mitokondrial şişme tahlil tetikledi.
Mitokondrial yalıtım için tüm malzeme ve tüpler buza, özellikle hücreleri homojenizasyon zaman olduğundan emin olun. % 0.25 tripsin-EDTA yemek 37 ° C'de 5 min için hücrelerden ayırmak için de kullanılabilir. 15-25 vuruş sonra mikroskop altında sağlam hücre zarı gözlemlemek ve mitokondri zar kırılma ö...

Mitokondri memeli hücrelerinde oksidatif fosforilasyon tarafından kullanılan ATP yaklaşık % 95'i üretmek için ana hücresel organlar vardır. Bu münzevi micromolar konsantrasyonu Ca2 + mitokondri tarafından ADP ve inorganik fosfat varlığı ADP sentez ATP1' e fazdan için kullanılabilir bildirilmektedir. Konsantrasyonu sitozolik Ca2 + bir eşiğin gittiğinde, mitokondri alımını Ca2 + hızla olabilir ve sızma bu yavaş yavaş. Böylece, işleyen mitokondri akını artan sitozolik Ca2 +olabilir. Oksidatif fosforilasyon katılımı için alakasız, mitokondrial Ca2 + Ayrıca sitozolik kalsiyum sinyaller de katılmak ve mitokondriyal iç mPTP açılış inducing tarafından mitokondrial apoptotik mekanizması harekete geçirmek membran2. Bu çok anormal yükselmesine intrasellüler Ca2 + 3mPTP açarak mitokondrial büyük şişlik neden olabilir kabul edilmiştir. Böylece, mitokondriyal işlev mitokondrial Ca2 + saklama kapasitesi ve Ca2 +miktarının tarafından değerlendirmek için bu protokolü amaçlamaktadır-mitokondrial şişlik trigged.
CA2 + saklama kapasitesi alımı sitozolik kalsiyum mitokondri yeteneği ölçümdür. Mitokondri sitozolik bedava kalsiyumu tampon ve kalsiyum-bağımlı hücresel süreçler tarafından etkin olmayan precipitates şeklinde kalsiyum alımını düzenleyen. Engelli Ca2 + saklama kapasitesi enerji sınırlama ve hatta nörodejeneratif hastalıklar4,5ile ilgili stres olayları oluşur. İzole mitokondri veya hücreleri digitonin permeabilized Ca2 + saklama kapasitesi tanımlamak için kullanılabilir ve Ca2 + ek Glutamat ve malat ile izole mitokondri tarafından birikir yeteneği yükseltilmiş tarafından etkilenen değil Mitokondrial yalıtım yordamı6. Digitonin titrated bir miktar farklı hücre tipleri plazma zarı permeabilize için kullanılmalıdır. Ca2 +hexapotassium tuz-yeşil flüoresan boya, bir Ca2 +bağlama-hassas cep impermeant görünür ışık telaşlı göstergesi, yaygın olarak kullanılan7oldu. CA2 +-floresan boya düşük-benzeşme göstergesidir ve hücre içi ücretsiz Ca2 + konsantrasyonları sırasında yükselmeye devam göstermek için kullanılan yeşil bağlama uzun süreli (5 dk) elektrodlar8. Bu yöntem radyoaktif filtre tekniği daha az hassas ancak büyük ölçüde basitleştirilmiştir. Ek Ca2 + kalsiyum yeşil Floresans yükseltir ve Ca2 + alımı mitokondri tarafından Floresans taban çizgisine döndürür. Mitokondri alımı extramitochondrial Ca2 + 9başarısız kadar Ca2 + sıralı eklemeler yapıldı. Bir floresan Mikroplaka okuyucu sürekli kalsiyum yeşil Floresans bildirmek için kullanılabilir.
Mitokondri depolarized Ca2 + birikimi sonra yayımlanan Ca2 + orta ve şişmeye başladı. CA2 +-şişme mPTP açılış belirtmek için kullanılır mitokondri tetikledi. Elektron mikroskobu ve Ca2 +ölçmek için kullanılan 540 nm can ışık Absorbans azalma-mitokondrial şişme10,11tetikledi. Mitokondri birim doğrudan nerede Absorbans düşüşler mitokondrial matris pasif şişmesi yansıtmak ileriye doğru açı ışık saçılma12' belirlenebilir.
Burada, mitokondrial Ca2 + saklama kapasitesi ve Ca2 +incelemek için metodoloji göstermek-SH-SY5Y hücreleri izole mitokondri mitokondrial şişme tetikledi.

<table><tbody><tr><td>SH-SY5Y cell line</td><td>ATCC (The Global Bioresource Center)</td><td>ATCC Number: CRL-2266</td><td></td></tr><tr><td>Calcium green-5N</td><td>Life Technologies</td><td>c-3737</td><td>Ca&lt;sup&gt;2+&lt;/sup&gt;-binding green fluorescent dye</td></tr><tr><td>complete protease inhibitors</td><td>Roche Molecular Biochemicals</td><td>4693116001</td><td></td></tr><tr><td>glass homogenizer</td><td>Kimble Chase</td><td>9885303002</td><td></td></tr><tr><td>glutamate</td><td>Sigma-Aldrich</td><td>RES5063G-A7</td><td></td></tr><tr><td>malate</td><td>Sigma-Aldrich</td><td>46940-U</td><td></td></tr><tr><td>rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>00457</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900050</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900266</td><td></td></tr><tr><td>Varioskan flash instruments</td><td>Thermo Scientific</td><td>IC100E</td><td></td></tr><tr><td>Bongkrekate</td><td>Biovision</td><td>1820-100</td><td></td></tr><tr><td>atractyloside</td><td>Sigma-Aldrich</td><td>C4992</td><td></td></tr><tr><td>high-glucose Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Hyclone</td><td>SH30022</td><td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td></tr><tr><td>fetal bovine serum</td><td>Hyclone</td><td>SV30087</td><td></td></tr><tr><td>penicillin</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>streptomycin</td><td>Invitrogen</td><td>11860-038</td><td></td></tr><tr><td>BCA assay kit</td><td>Thermo Scientific</td><td>NCI3225CH</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>NEST</td><td>704001</td><td></td></tr></tbody></table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

ATP üretimi oksidatif fosforilasyon tarafından mitokondri birincil işlevidir. Mitokondri daha yüksek ökaryotlarda da tampon sitozolik Ca2 + içinde katılmak ve mitokondriyal ATP üretiminde intramitochondrial ücretsiz Ca2 + konsantrasyon tarafından aracılık. CA2 + saklama kapasitesi mitokondri kalsiyum mitokondrial matris korumak yeteneği olarak kabul edilebilir. Birikmiş intrasellüler Ca2 + yol açan iç mitokondrial membran geçirgenliği molekülleri ile bir moleküler kaçağı neden daha az 1,5 kDa ağırlık mitokondrial geçirgenliği geçiş gözenek (mPTP), açılış olarak adlandırdığı. CA2 +-şişme mPTP açılış belirtmek için kullanılır mitokondri tetikledi. Burada, biz Ca2 + saklama kapasitesi ve Ca2 +incelemek için iki deneyleri tarif-izole mitokondri mitokondrial şişme tetikledi. Ca2 + eklenir belirli miktarda sonra tüm adımları bir günde tamamladı ve Mikroplaka okuyucu tarafından kaydedildi. Böylece, bu iki basit ve etkili deneyleri Ca2 +değerlendirmek için kabul edilebilir-mitokondrial işlevleri ile ilgili.

Ca2 + saklama kapasitesi temsilcisi sonuçları: Sonuçlar floresan değerler olarak ifade edildi. Ve Ca2 + (200 nmols/mg mitokondrial protein), yukarıda mPTP açılış temelle Kişilik artışla Floresans ek darbeleri ile. 5 mikron Bongkrekate (BKA), bir inhibitörü olan Ca2 +-indüklenen mPTP açılış veya 1 mikron atractyloside (ATR), Ca2 +bir aktivatör-mPTP açılış indüklenen, izole mitokondri içinde eklenmiştir. Mikt...

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Mitokondrial Ca2 + saklama kapasitesi tahlil ve Ca2 +-mitokondrial şişme tahlil tetiklenen

Ca2 + saklama kapasitesi ve Ca2 +incelemek için bir yöntemi açıklamak için bu protokolü amaçlamaktadır - mitokondrial SH-SY5Y izole mitokondri şişme adım adım hücreleri tetikledi.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

mitochondrial-ca2-retention-capacity-assay-ca2-triggered

Research

JoVE Journal

Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

11.1K Görüntüleme.  Qilu Hospital of Shandong University. Ca2 + saklama kapasitesi ve Ca2 +incelemek için bir yöntemi açıklamak için bu protokolü amaçlamaktadır - mitokondrial SH-SY5Y izole mitokondri şişme adım adım hücreleri tetikledi.

Video: Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Edu Sinyal Amplifikasyon Bireysel Hücrelerde Mitokondriyal DNA replikasyonu Görselleştirme

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Nörobilim

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

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

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

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

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

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitokondrial Membran Potansiyeli ve Reaktif Oksijen Türleri Canlı Rat Kortikal nöronlar Belirlenmesi

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Mitokondrial kalsiyum akını izole mitokondri ve kültürlü hücreleri analizleri

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Biyokimya

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, ...

Biyoloji

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Floresan Mikroskopi Canlı hücreler Potansiyel Mitokondriyal Kalsiyum ve Mitokondriyal Membran Eşzamanlı Ölçümü

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

Kültürlenmiş Hücrelerde Mitokondriyal Kalsiyum Alımını Ölçmek için Kalsiyum Akışı Testi

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

JoVE Encyclopedia of Experiments

Biyolojik Teknikler

Tahlil Teknikleri

Hücre bazlı analizler

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Mitokondriyal Kalsiyum Alım Testi: İzole Mitokondride Kalsiyum Alımını Ölçmek İçin Plaka Okuyucu Tabanlı Bir Yöntem

Biyokimyasal analizler

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
This video ...

Mitokondrial Ca^{2 +} saklama kapasitesi tahlil ve Ca^{2 +}-mitokondrial şişme tahlil tetiklenen

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler