Biz sitometresi akış erişimi için Laurie Destroismaisons ve üstün teknik destek için Sarah PEYRARD ve Dr.Alexandre Prat teşekkür ederiz. Biz de hazırlıkları miyelin kaldırılması ile ilgili yaptığı katkılarından dolayı Dr Timothy Miller kabul etmek istiyorum. Bu çalışma Sağlık Araştırması (CIHR) Nöromusküler Araştırma Ortaklığı, Yenilik için Kanada Vakfı, Kanada ALS Derneği, ALS Araştırma Frick Vakfı, CHUM Vakfı ve Fonds de la Recherche en Santé du Québec (CVV) olan Kanada Enstitüleri tarafından desteklenmiştir. Hem CVV ve NA olan Araştırma Âlimler Fonds de la Recherche en Santé du Québec ve CIHR Yeni Müfettişler. SP Kanada ALS Derneği&#39;nin Tim Noël Öğrencilik tarafından desteklenmektedir.

Bu çalışmada kullanılan hayvanların Kanada tarafından belirtildiği gibi ulusal standartlar aşağıdaki Hayvanları Koruma Centre de Recherche du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM) Kurumsal Komitesi tarafından onaylanmış bir protokol (N08001CVsr) sıkı göre tedavi edildi Hayvan Bakım Konseyi (ccac). Bu protokolü (Tablo 1) gerçekleştirmek için gerekli tüm reaktifleri hazırlayın. Ekipman, malzeme ve tedarikçi ile ilgili diğer tüm detayları Malzeme Listesi bulunabilir. <img alt="Tablo 1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> Tablo 1. Tampon bileşimler. Sıçan Omurilik 1. Koleksiyonu <ol><li> Derinden% 4 isofloran ile sıçan (Sprague Dawley) uyuşturan. Th sıkıştığı üzerine refleksi eksikliği ile anestezi doğrulayıne forepaw. Giyotin ile dekapitasyon yoluyla sıçan öldürülür. Ötenazi Bu yöntem omuriliği deforme olabilir başkaları üzerinde tercih edilir. </li><li> Omurga ortaya çıkarmak için arka cilt kesin. Sadece leğen kemiği üzerinde kemik makası ile omuriliğe kesin. Vertebral kolonun açılmasını gözünüzde canlandırın. </li><li> Vertebral kolona, ​​Fosfat Tamponlu Salin (PBS) ile doldurulmuş bir 200 ul pipet ucu (alev biraz üzerinde eriyen yoluyla bağlanmış) ile, bir 10 ml şırınga yerleştirin. </li><li> Pistonun basınç orta tatbik edilmek sureti ile omurilik yıkayın. </li><li> Herhangi bir kan, omurilik mevcut ise, bir sonraki aşamaya geçmeden önce PBS ile yıkayın. NOT: Bu yöntem aynı zamanda beyin ve karaciğer için valide edilmiştir. Bu dokular dahil olmak üzere, durumunda, yarısı kurban sıçandan beyin ve omurilik ağırlıkça eşit bir karaciğer parçası toplar. Diğer tüm adımlar aynıdır kalır. </li></ol> Omurilik Mit 2. izolasyonuochondria (Vande Velde ve ark uyarlanmıştır. 4) <ol><li> 5 hacim (~ 3.25 mi) Homojenleştirme tamponu (HB) ile 5 ml&#39;lik bir cam homojenizatör içinde, bütün dokunulmamış omurilik yeri ve toplayın. İzole mitokondri optimal kurtarma için, buz veya soğuk odada tüm adımları uygulayın. Doku hiçbir büyük parça kalmayıncaya kadar, elle yaklaşık sekiz vuruşları doku homojenize. İki (2 mi) ve üç (1.7 mi) mikrosantrifüj tüplerinde Homojenat yerleştirin. Bir tezgah üstü mikrosantrfuj, 4 ° C&#39;de 10 dakika 1,300 x g santrifüj. </li><li> 5 ml&#39;lik bir ultrasantrifüjdeki tüpüne süpernatant ve yer kurtarın. Pelet içeren mikrosantrifüj tüp 750 ul (~ 0.5 hacimler) HB ekleyin ve hafifçe pelletini. Tekrar santrifüj ve yeniden süspansiyona iki kez daha yineleyin. Havuz yukarıdaki ile aynı 5 ml&#39;lik bir ultra santrifüj tüpüne tüm üst sıvıları (S1a ve S1 B). Bu adım, küçük enkaz kaldırmak için hizmet vermektedir. </li><li> Santrifüj S1 sallanan bir kova çürüklüğü ile donatılmış bir ultrasantrifüjdeki kullanılarak toplandıya da 4 ° C&#39;de 15 dakika boyunca 17,000 x g&#39;de santrifüjleyin. </li><li> Sitosolik parçacık, (Western blot analizi için örneğin) ilgi ise daha sonraki işlemler için bir süpernatan (S2) muhafaza edilmelidir. 4 mi HB + 50 mM KCI pelet (P2) ham mitokondriyal fraksiyonu, yeniden süspanse edin. Bir sallanan kepçeli rotor içinde 4 ° C&#39;de 15 dakika 17,000 x g santrifüj. Süpernatantı atın ve yavaşça 800 ul HB pelet (P3) tekrar süspansiyon. Not: Mitokondri herhangi bir özel olmayan mitokondriyal ilişkili kirleri çıkarmak HB ile + 50 mM KCI ile yıkanmıştır. </li><li> Yeni 5 ml&#39;lik bir ultra santrifüj tüp içinde, süspansiyon haline getirilen tanelere, (P3) tam olarak 800 ul ekleyin. </li><li> Bu tüp, böylece% 12 iyodiksanol bir nihai konsantrasyonu yaratırken, iyodiksanol (yoğunluk gradyan ortamı) 200 ul ilave edin. Yavaşça ancak iyice P1000 ile pipetleme yoluyla tüpün içindekiler karıştırılır. İlk iyodiksanol eklenmesi ve daha sonra tekrar askıda topak (P3) iyice karışmasını kolaylaştırmak için tercih edilir. Bir Ult yılında santrifüj4 ° C&#39;de 15 dakika boyunca 17,000 x g&#39;de bir sallanan kepçeli rotor ile donatılmıştır racentrifuge. </li><li> Not: Karaciğer miyelin içermeyen ve sadece karaciğer işlenirken, bu nedenle, bu adım gerekli değildir. Karaciğer MSS mitokondri ile eş zamanlı olarak, işlenmekte olan, ancak eşit tüm dokularını tedavi etmek için önerilmektedir. </li><li> Tüpün üst miyelin tabakasını aspire ve dikkatli bir şekilde çıkarın ve süpernatantı atın. Pelet gevşek olabilir. 4 ml HB pelletini. Tekrar santrifüj 4 ° C&#39;de 10 dakika boyunca 17,000 x g&#39;de. Süpernatantı atın ve 4 ml HB pelletini. Santrifüj tekrarlayın ve süpernatant kaldırmak. </li><li> 100-200 ul HB nihai pelet (P7) tekrar ve bir 1.7 ml mikrosantrifüj tüp transfer. Bu örnek izole mitokondri içerir. </li><li> Protein miktar tayini geçin. Mitokondri pahalı ne yeterli çözülmesini sağlamak için örnekler ve% 2 sodyum dodesil sülfat (SDS) standart eğri seyreltinng protein ölçümü. </li></ol> Akış Sitometrinin için İzole Mitokondri 3. ile immün <ol><li> Her boyama karışımı test edilebilmesi için, pipet 1,7 ml mikrosantrifüj tüp içine izole mitokondri 25 mcg. Her deneyde bir boyanmamış örnek sayılabilir; ve her bir antikor için, uygun izotip kontrolü için bir örnek eklemeyi unutmayın. </li><li> Bir tezgah üstü mikrosantrifüj 4 ° C&#39;de 2 dakika boyunca 17,000 x g&#39;de santrifüjleyin. </li><li> Süpernatantı ve 4 ° C (bloke etme adımı) &#39;de 15 dakika boyunca% 10-yağ asitsiz BSA ile takviye edilmiş 50 ul mitokondri tamponu (Tampon E) olarak izole mitokondri tekrar süspansiyon. Not: etiketleme sırasında, 4 ° C&#39;de bir buzdolabı içinde inkubasyon gerçekleştirmek. </li><li> Tüpüne birincil antikor (tavşan anti-Mfn2, ml başına 20 ug) ilave edildi ve 4 ° C&#39;de 30 dakika inkübe edilir. NOT: titrasyonu ile ampirik olarak her antikor optimal konsantrasyonunu belirleyin. Nedeniyle konsantrasyon ve / veya p değişkenliğeurity, aynı üreticinin aynı antikorun farklı partiler, farklı sonuçlara yol açabilir; Dolayısıyla antikor titrasyonu her yeni lot için gereklidir. </li><li> Bağlanmamış birinci antikorları yıkayın: Santrifüj 17,000 x g&#39;de 2 dakika süre ile 4 ° C &#39;de. Süpernatantı ve yavaşça 200 ul M Tampon pelletini. 4 ° C&#39;de 2 dakika boyunca 17,000 x g&#39;de santrifüjleyin. Süpernatantı ve 50 ul M Tampon pelletini. </li><li> Tüpüne ikincil antikoru (Eşek anti-tavşan IgG Fikoeritrin (PE), ml başına 0.5 ug) ilave edilir ve ışıksız bir ortamda 4 ° C sıcaklıkta 30 dakika boyunca inkübe örnekleri. </li><li> Bağlanmamış ikinci antikorları yıkayın: Santrifüj 17,000 x g&#39;de 2 dakika süre ile 4 ° C &#39;de. Süpernatantı ve 200 ul M Tampon pelletini. 4 ° C&#39;de 2 dakika boyunca 17,000 x g&#39;de santrifüjleyin. Süpernatantı ve 500 ul M Tampon pelletini. </li><li> Olaylar aslında mitokondri içinde emin olmak için, bir ile leke izole mitokondriışıksız bir ortamda oda sıcaklığında 15 dakika boyunca mitokondri özel floresan boya. Diğer fonksiyonel parametreler (mitokondriyal transmembran potansiyeli veya süperoksit üretimi) boyama isteniyorsa, değilse, edinimi için 5. adıma 4. adıma geçin. NOT: Bu ikincil antikor emisyon spektrumu fonksiyonel boyaların olduğu ile uyumlu olduğunu doğrulamak için önemlidir. (: Örnek 650 nm / Em 660 nm APC), örneğin, tetrametilrodamin metil ester (TMRM) FITC ve zar potansiyeline benzer spektral özelliklere sahip ticari bir boya ile mitokondriyal saflık doğrulanması durumunda, uygun bir ikincil antikor alofikosiyanin olacaktır. Tazminatlar kontrolleri ekleyin, yani bir örnek immunolabeled veya uygulanabilir olduğunda tek bir floroforla ile boyandı. </li><li> Sitometresiyle yükleme akışı için uygun bir tüp aktarın. (. Küçük örneklem büyüklüğü bir mikro tüp ml&#39;lik bir tüp akış sitometresini 3 içine yerleştirilir kolaylaştırmak için) buz üzerinde örnekleri tutun ve hemen devamedinimi için akış sitometresi. </li></ol> 4. Assaying Mitokondriyal Transmemebran Potansiyeli ve Sitometrisi ile Mitokondriyal süperoksit Üretimi <ol><li> Izole edilmiş mitokondri ışıktan koruyarak oda sıcaklığında 15 dakika boyunca aşama 3.8 ile 100 nM TMRM (Örnek 548 nm / Em 574 nm): 5 ile lekeleme ile bir dokunulmamış zar potansiyeline sahip olduğundan emin olun. Örnekler ve popülasyonlar, TMRM (1-30 nM) &#39;nin alt / olmayan söndürme konsantrasyonlarının kullanımı arasındaki zar potansiyeli Karşılaştırma için, 6 daha uygun olabilir. <ol><li> TMRM boyama, 100 uM karbonil siyanit m-kloro fenil hidrazin (CCCP) varlığında 100 nM TMRM boyanan izole mitokondri, mitokondri depolarize bir mitokondriyal uncoupler için bir kontrol olarak. Boyanın daha düşük konsantrasyonlar kullanıldığı takdirde mitokondri depolarize için gerekli CCCP konsantrasyonu daha az olabilir. </li></ol></li><li> İzole mitokondri st tarafından mitokondriyal süperoksit üretmek doğrulayınışıksız bir ortamda oda sıcaklığında 15 dakika boyunca, aynı zamanda adım 3.8 değerinde, uygun bir mitokondriyal süperoksit gösterge 7, geri kalan. <ol><li> Mitokondriyal süperoksit üretimi, 10 uM Antimycin A, mitokondriyal süperoksit üretimini artıracak solunum zinciri kompleksi III bir inhibitörün mevcudiyetinde boya ile leke izole mitokondri için bir kontrol olarak. </li></ol></li></ol> Sitometrisi ile kondriyayı İzole immunolabeled 5. Toplama ve Analiz <ol><li> Enstrüman kurmak: doğrusal geçiş gerilimleri izole mitokondri ve set gerilimlerin analizi kolaylaştırmak için log modu için (FSC: 450; SSC: 250). Olaylar FSC-A (alan) modu yanı sıra FSC-W (genişlik) ve FSC-H (yükseklik) toplanan emin olun, ikilileri (dışlamak mümkün yani, aynı zamanda dedektör geçen iki olay, ) analizi sonrası veri toplama. Olayların Set sayısı 100,000 ile tahsil edilecek. Tazminat kontrolleri EdinmeVarsa. </li><li> Veri toplama: veri toplama önce numune vorteks kaçının. Bunun yerine hafifçe tüp dokunarak karıştırın. Başlangıçta gating sırasında düşük bir basınçta olay toplamak. Toplam nüfus Kapısı. , Buna göre histogramlarının gerilimler ayarlayın genellikle boyanmamış numunenin tepe ikinci on (10 2) karşılık gelir. Kapılar kurulmuş, ve örnekler işleniyor sonra, basıncı yüksek geçirilebilir. </li><li> Analiz: FSC-W FSC karşı komplo tarafından ikilileri gözünüzde canlandırın. Singletlerini ve ikilileri tanımlayın. Singletlerin üzerinde Kapısı. Mitokondriyal-selektif boya ile pozitif boyandı olaylar kapılayarak mitokondriyal nüfusu seçin. </li><li> Izotop kontrolden plan etiketleme belirleyin. Izotip kontrol örneği kullanarak, Mfn2 antikor için pozitif popülasyon mitokondriyal etiketleme yüzdesini belirlemek. </li></ol>

<ol>
	<li>Itoh, K., Nakamura, K., Iijima, M., Sesaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+in+neurodegeneration.">Mitochondrial dynamics in neurodegeneration.</a> Trends Cell Biol. 23, 64-71 (2013).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+is+recruited+selectively+to+impaired+mitochondria+and+promotes+their+autophagy.">Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.</a> J Cell Biol. 183, 795-803 (2008).</li><li>Zorov, D. B., Kobrinsky, E., Juhaszova, M., Sollott, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+intracellular+organelle+function+using+fluorescent+probes:+from+animalcules+to+quantum+dots.">Examining intracellular organelle function using fluorescent probes: from animalcules to quantum dots.</a> Circ Res. 95, 239-252 (2004).</li><li>Vande Velde, C., Miller, T., Cashman, N. R., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+association+of+misfolded+ALS-linked+mutant+SOD1+with+the+cytoplasmic+face+of+mitochondria.">Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria.</a> Proc Natl Acad Sci U S A. 105, 4022-4027 (2008).</li><li>Loew, L. M., Tuft, R. A., Carrington, W., Fay, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+five+dimensions:+time-dependent+membrane+potentials+in+individual+mitochondria.">Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.</a> Biophys J. 65, 2396-2407 (1993).</li><li>Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+probes+and+the+proton+gradient:+a+practical+usage+guide.">Mitochondrial membrane potential probes and the proton gradient: a practical usage guide.</a> Biotechniques. 50, 98-115 (2011).</li><li>Xu, X., Arriaga, 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=Qualitative+determination+of+superoxide+release+at+both+sides+of+the+mitochondrial+inner+membrane+by+capillary+electrophoretic+analysis+of+the+oxidation+products+of+triphenylphosphonium+hydroethidine.">Qualitative determination of superoxide release at both sides of the mitochondrial inner membrane by capillary electrophoretic analysis of the oxidation products of triphenylphosphonium hydroethidine.</a> Free Radic Biol Med. 46, 905-913 (2009).</li><li>Otera, H., Ishihara, N., Mihara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+function+and+regulation+of+mitochondrial+fission.">New insights into the function and regulation of mitochondrial fission.</a> Biochim Biophys Acta. 1833, 1256-1268 (2013).</li><li>de Brito, O. M., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitofusin+2+tethers+endoplasmic+reticulum+to+mitochondria.">Mitofusin 2 tethers endoplasmic reticulum to mitochondria.</a> Nature. 456, 605-610 (2008).</li><li>Wikstrom, J. D., Twig, G., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+mitochondrial+heterogeneity+tell+us+about+mitochondrial+dynamics+and+autophagy.">What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy.</a> Int J Biochem Cell Biol. 41, 1914-1927 (2009).</li><li>Pickles, S., Destroismaisons, L., Peyrard, S. L., Cadot, S., Rouleau, G. A., Brown, R. H., Julien, J. P., Arbour, N., Vande Velde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+damage+revealed+by+immunoselection+for+ALS-linked+misfolded+SOD1.">Mitochondrial damage revealed by immunoselection for ALS-linked misfolded SOD1.</a> Hum Mol Genet. 22, 3947-3959 (2013).</li><li>Frank, S., Gaume, B., Bergmann-Leitner, E. S., Leitner, W. W., Robert, E. G., Catez, F., Smith, C. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dynamin-related+protein+1,+a+mediator+of+mitochondrial+fission,+in+apoptosis.">The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.</a> Dev Cell. 1, 515-525 (2001).</li><li>West, A. P., Brodsky, I. E., Rahner, C., Woo, D. K., Erdjument-Bromage, H., Tempst, P., Walsh, M. C., Choi, Y., Shadel, G. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+signalling+augments+macrophage+bactericidal+activity+through+mitochondrial+ROS.">TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.</a> Nature. 472, 476-480 (2011).</li><li>Abad, M. F., Di Benedetto, G., Magalh&#227;es, P. J., Filippin, L., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pH+monitored+by+a+new+engineered+green+fluorescent+protein+mutant.">Mitochondrial pH monitored by a new engineered green fluorescent protein mutant.</a> J Biol Chem. 279, 11521-11529 (2004).</li><li>Murphy, A. N., Bredesen, D., 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, 9893-9898 (1996).</li><li>Tantama, M., Martinez-Fran&#231;ois, J. R., Mongeon, R., Yellen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+energy+status+in+live+cells+with+a+fluorescent+biosensor+of+the+intracellular+ATP-to-ADP+ratio.">Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio.</a> Nat Commun. 4, 2550 (2013).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu mitokondri normal fizyoloji ve hastalık hem de kilit oyuncular olduğu giderek açıktır. Imünoblotting, belli bir durumda mitokondri içinde veya mitokondriyal yüzeyinde tüm nüfusun ortalama bu yöntem raporlar bulundu hangi proteinlerdir çıkarabilirler. Bu yöntem mitokondriyal alt popülasyonlarının veya altkümelerin göreceli bolluk hakkında bilgi elde edemez. O olmuştur iken daha önce tüm mitokondri eşit, alan giderek bir hücre içinde bu mitokondri kabulüdür oluşturulan morfolojisi ve / veya...

Mitokondri, yüksek enerji talebinin sitelerine taşınan ve fizyolojik uyaranlara 1 hızla yanıt olan, fizyon ve füzyon birden fazla tur geçmesi son derece dinamik organellerdir. Giderek farklı dokularda olduğu mitokondri kabul edildiğinden, hatta farklı hücre bölmeleri, yeni yöntemler bu farklı mitokondriyal alt kümelerini tanımlamak için gerekli olan, farklı fonksiyonel profilleri var. Mikroskopi bireysel mitokondri görselleştirilebilir sağlayan bir aracı ve bağışıklık 2 ile belirlenebilir veya mitokondrilerde bir proteinin varlığını içerir. Bununla birlikte, bu yöntem ile kantitatif analiz, emek yoğun ve ölümsüzleştirilmiş veya primer hücre çizgileri kullanılarak deneyler için daha uygundur. Dokusundan türetilen bireysel mitokondri çalışması çok daha zordur ve çok yöntem ile aynı zamanda mitokondriyal değerlendirmeleri mevcuttur alt gruplarının kolay tanımlama için izin vermezmitokondriyal fonksiyon 3 tirme. Bu engel, akış sitometrisi ile kemirgen dokulardan izole edildi ve daha sonra analiz mitokondri immunolabel için yeni bir metot gidermek amacıyla geliştirilmiştir. Bu, hızlı bir şekilde saptanması ve mikroskobik inceleme ile karşılaştırıldığında mitokondriyal dış membran, lokalize proteinler ölçülmesini sağlar, daha az emek yoğun ve tek bir numunede bulunan mitokondri binlerce analiz edilmesini sağlar. Bu deney, mitokondri de yapısal olarak mevcut olduğu düşünülmektedir mitokondriyal dış zar proteinlerinin &#39;nin ve nispi miktarını izlemek için uygulanabilir, mitokondriyal yüzeyine proteinler işe ya da patolojik koşulların mitokondrilere mislocalized proteinlerin tespiti. Ayrıca, geleneksel floresan gösterge boyaların kuruluş farklı mitokondriyal mitokondriyal fonksiyonun bazı yönlerinin aynı anda değerlendirilmesini sağlarsubpopülasyonunun.

<table border="0" cellpadding="0" cellspacing="0" width="1276">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:349px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:543px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Rats</td>
			<td style="width:349px;">Charles River</td>
			<td style="width:119px;">Strain code 400</td>
			<td style="width:543px;">Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dounce homogenizer</td>
			<td style="width:349px;">Kontes Glass Co.&#160;</td>
			<td style="width:119px;">885450-0022</td>
			<td>Duall 22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Microcentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall Legend Micro 17 R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ulara-Clear Ultracentrifuge tubes</td>
			<td style="width:349px;">Beckman Coultier</td>
			<td style="width:119px;">344057</td>
			<td>Transparent, thin walled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sorvall Ultracentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall WX UltraSeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">AH-650 rotor and buckets&#160;&#160;</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">Opti-prep</td>
			<td style="width:349px;">Axis-Shield</td>
			<td style="width:119px;">1114542</td>
			<td>Iodixanol, density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fatty acid free Bovine Serum Albumin</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8806</td>
			<td>Must be fatty acid free for mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sodium succinate dibasic hexahydrate&#160;&#160;</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">S9637</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:267px;">Rabbit anti-Mitofusin2 antibody</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">M6319</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:267px;">Rabbit IgG</td>
			<td style="width:349px;">Jackson Immuno Research&#160;</td>
			<td style="width:119px;">011-000-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Anti-Rabbit IgG PE</td>
			<td style="width:349px;">eBioscience</td>
			<td style="width:119px;">12-4739-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Micro titer tube</td>
			<td style="width:349px;">Bio-Rad</td>
			<td style="width:119px;">223-9391</td>
			<td>For sample acquisition by flow cytometry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoTrackerGreen (MTG)</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M7514</td>
			<td>100 nM: Ex490 nm/Em516 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">TMRM</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">T668</td>
			<td>100 nM: Ex548 nm/Em574 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CCCP</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoSOX Red</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M36008</td>
			<td>5 &#181;M: Ex540 nm/Em600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Antimycin A</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8874</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">LSR II flow cytometer</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BS FACSDiva Software</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FlowJo</td>
			<td style="width:349px;">TreeStar Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Software used for analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">BCA protein assay kit&#160;&#160;</td>
			<td style="width:349px;">Pierce/Thermo Scientific</td>
			<td style="width:119px;">23225</td>
			<td style="width:543px;">Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td>
		</tr>
	</tbody>
</table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

Yöntem algılamak ve hayvansal dokular mitokondriyal fizyolojisi ve patofizyolojisi çalışma için hayati olan mitokondriyal dış membran proteini bileşenleri izlemek için. Bu protokol kemirgen dokusundan izole mitokondri immunolabeled ve flow sitometri ile analiz edilir bir yöntem açıklanır. Mitokondri kemirgen omurilik izole edildi ve miyelin, sinir dokusundan hazırlanan mitokondriyal parçaların büyük bir kirletici maddeyi bertaraf edilmesi için hızlı bir zenginleştirme adımına tabi tutulurlar. İzole mitokondri daha sonra tercih edilen bir antikoru ve bir floresan konjuge sekonder antikor ile etiketlenir. Akış sitometrisi ile analiz, algılama ve imüno-işaretli protein miktarının ölçülmesi ile takip mitokondriyal belirli bir boya ile boyanması ile mitokondriyal preparatların saflığı göreli doğrular. Bu teknik, numune başına mitokondri yüzbinlerce analizine izin veren, hızlı ölçülebilir ve yüksek verimli olduğunu. Bu yeni p değerlendirmek için geçerlidirNormal fizyolojik şartlar altında mitokondrial yüzeyinde roteinler gibi patoloji sırasında bu organele mislocalized olabilir proteinleri. Önemli olarak, bu yöntem, alt-popülasyonlarının mitokondriyal bazı etkinlikler rapor floresan göstergesi boyalar ile birleştirilebilir ve merkezi sinir sistemi (beyin ve omurilik) yanı sıra karaciğer, mitokondri için mümkün olduğunu edilebilir.

Sıçan omurilik türetilen mitokondri Mitofusin2 (Mfn2), mitokondri 8 dış membran füzyon rol oynayan bir protein hedefli bir antikor ile imüno edilebilir. Bir Mfn2 spesifik antikor ve bir floresan konjuge sekonder antikor ile izole edilmesini ve ardından etiketleme, mitokondri akış sitometrisi (Şekil 1) tarafından işlenir. Veri toplama sonra, numuneler ilk olarak, bir nokta arsa (Şekil 2A) ilgili toplanan tüm olayları görselleştirerek, analiz yazılımı akı?...

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Burada açıklanan tespit ve akış sitometrisi ile analiz kemirgen doku ve izole mitokondri immün ile mitokondriyal dış membran proteinleri ölçmek için bir yöntemdir. Bu yöntem, mitokondriyal alt popülasyonlarının fonksiyonel özelliklerini değerlendirmek için genişletilebilir.

İzole Mitokondri Akış Sitometrisi ile Dış Zar Proteinleri imüno

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Research

JoVE Journal

Biology

15.4K Görüntüleme.  Université de Montréal, CRCHUM.  Burada açıklanan tespit ve akış sitometrisi ile analiz kemirgen doku ve izole mitokondri immün ile mitokondriyal dış membran proteinleri ölçmek için bir yöntemdir. Bu yöntem, mitokondriyal alt popülasyonlarının fonksiyonel özelliklerini değerlendirmek için genişletilebilir. 

Video: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP FCM: Sitometrisi ile Algılandı Immunoprecipitation

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Biyoloji

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Akış Sitometri Plazma Membran GLUT4 Translokasyon Kantitatif Ölçüm

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Fare Mayotik Hücreler Sitometrisi Arıtma

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Cep Takip Boyalar kullanarak Hücre Bölünmesi İzleme için optimize Boyama ve Çoğalma Modelleme Yöntemleri

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Sitometrisi ile Böbrek Hücrelerine sitozolik kalsiyum Ölçümleri

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

MitoCeption: Glioblastoma Kök Hücreleri için İzole İnsan MSC kondriyayı aktarma

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Spektral Sitometrisi ile Katı Dokular İzole Hücre Cezalar Analizi

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...