Bu çalışma Eagles İnovasyon Ödülü (Eagles Kardeş Sipariş) ve Mayo Vakfı tarafından desteklenmiştir.

Yerli protein-protein etkileşimleri ve akış sitometri multiprotein kompleksleri Yüksek hassasiyet algılama ve kantitatif analiz: ** Bu video protokolü ilişkili bir yayın 1 dayanmaktadır. Adam G. Schrum, Diana Gil, Elaine P. Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang WA Schamel Ed Palmer. Bilim STKE 2007 (389): PL2 5 Haziran, 2007, [DOI: 10.1126/stke.3892007pl2] <a href="http://stke.sciencemag.org/cgi/content/abstract/2007/389/pl2">Bu yayının görmek için buraya tıklayınız</a> . Başlamadan Önce, ardından Sulu Stok Çözümleri hazırlayın: <table><tbody><tr><td> MES Kaplin Tampon: </td><td> Mağaza 25 ° C </td></tr><tr><td> MES, pH 6.0 </td><td> 50 mM </td></tr><tr><td> EDTA </td><td> 1 mM </td></tr><tr><td> Su Verme / / Depolama (QBS) Tampon Engelleme: </td><td> Mağaza, 4 ° C </td></tr><tr><td> BSA </td><td> % 1 </td></tr><tr><td> Sodyum azid </td><td> % 0,02 </td></tr><tr><td> PBS, pH 7.4 </td><td> 1x </td></tr><tr><td> FCM Tampon: </td><td> Mağaza, 4 ° C </td></tr><tr><td> Tris, pH: 7.4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 100 mM </td></tr><tr><td> Sodyum azid </td><td> % 0,02 </td></tr><tr><td> FBS </td><td> % 5 </td></tr><tr><td> PBS, pH 7.4 </td><td> 1x </td></tr></tbody></table> Kullanım hemen önce hazırlayın: <table><tbody><tr><td> EDAC-MES: </td><td> MES Kaplin Tampon 50 mg / ml EDAC tozu çözülür </td></tr><tr><td> Digitonin solüsyonu (% 2 w / v): </td><td> 95 ısıtarak dH 2 O digitonin toz çözülür ° C 5min sonra buz üzerinde soğutma </td></tr><tr><td> Lizis Tampon: </td><td></td></tr><tr><td> Tris, pH: 7.4 </td><td> 50 mM </td></tr><tr><td> NaCl </td><td> 150 mm </td></tr><tr><td> Digitonin çözüm </td><td> % 1 </td></tr><tr><td> Proteaz inhibitörleri </td><td> 1x </td></tr><tr><td> Buz üzerinde saklayın </td><td></td></tr></tbody></table> 1. Boncuk için mAb çiftlenmesi <ol><li> PBS içinde seyreltilmesi ve bir hemasitometre sayım tarafından satın alınan stok boncuk konsantrasyon belirleyin. Sayımı için boncuk 1:10000 seyreltme ile başlayın. </li><li> Pipet 18 x 1.5 ml mikrosantrifüj tüpe 10 6 boncuk. </li><li> Her yıkamadan sonra, 25 3 dakika 20.000 g ° C santrifüj, 0,5 - 1,0 ml MES Kaplin Tampon boncuk, 2 ya da 3 kere yıkayın. </li><li> 50 mcL MES Kaplin Tampon boncuklar tekrar </li><li> Taze hazırlanmış EDAC-MES 20 mcL ekleyerek boncuklar karboksil grubu etkinleştirin. </li><li> , 25 ° C&#39;de elle yukarı ve aşağı pipetleme 15 dakika süreyle hafifçe karıştırın. </li><li> 25 3 dakika 20.000 g de santrifüj ° C Her yıkamadan sonra, 0,5 ila 1,0 ml PBS aktive boncuk, 2 ya da 3 kere yıkayın. </li><li> 50 mcL PBS içinde aktif boncuk süspanse edin. </li><li> Aktive boncuk çözüm IP mAb (0,2-1,0 mg / ml stok konsantrasyonu) 50 mcL ekleyin. </li><li> 25 ° C&#39;de 3 ila 4 saat titreşimli çalkalayıcı tüp koyarak karıştırın. Tüpün alt boncuk yerleşme önlemek için yeterince çalkalayınız. </li><li> Yıkama mAb birleştiğinde boncuklar her yıkamadan sonra 0,5 - 1,0 mL PBS içinde 2 - 3 kez, 3 dakika 25 ° C 20.000 g de santrifüj. </li><li> 100 mcL QBS Tampon boncuklar tekrar Bu 4 ° C&#39;de saklanabilir </li><li> Boncuk askıya iyi, PBS içinde 1:200-1:10,000 sulandırmak ve bir hemasitometre ile saymak. Konsantrasyonu her IP veya pull-down deneyde kullanılan boncuk sayısı tam olarak kontrol altına alınabilir, böylece, her preparatif parti için ölçülebilir olmalıdır. </li></ol> 2. Post-nükleer Lizat Hazırlama ve Ip Lizis yöntemi ve optimal lizis koşulları, hücre tipi ve protein-protein etkileşimleri incelenmektedir bağlıdır. Protokolleri bir dizi var ve uygulama için modifiye edilebilir. <ol><li> Lenfositler, veya 5 gibi 20 x 10 6 &#39;küçük&#39; hücreleri, Lyse x 10 makrofajlar ya da tümör hücrelerinin gibi 6 &#39;büyük&#39; ​​hücreleri, buz üzerinde 20 dk için 1.5 mL mikrosantrifüj tüp 100 mcL taze Lizis tampon . Gerektiği gibi lizis hacmi Ölçeği. </li><li> Çekirdek ve çözünmez hücresel enkaz kaldırmak için, 20.000 g de 10 dakika 4 ° C lizat santrifüj Süpernatantı tutun ve pelet iptal. </li><li> Karıştırmak için hafifçe pipetleme 0.5 x 10 açıklık lizat 5 ila 2,5 x 10 5 mAb çiftli boncuk, ekleyin. Minimum hacim için kullanabileceğiniz5 mcL tek bir IP gerçekleştirmek. </li><li> Dikey dönen bir tekerlek 4 saat gecede soğuk bir odaya yerleştirin. Boncuk yerleşmesini önlemek için yeterli hızına ayarlayın. Bu dönüşü sırasında tüpün alt kalmaya, düşük hacimli IP sıvı için kabul edilebilir. </li></ol> 3. Boncuk çekilen Protein Florokrom konjuge antikorlar ile problama <ol><li> 4 20.000 g santrifüj ° C Her yıkamadan sonra 3 dakika, 0,2 ila 1,0 mL buz FCM Tampon IP boncuk iki kez yıkayın. </li><li> Beş veya daha fazla bir baca alt plakası 1.5 mL mikrosantrifüj tüpler veya kuyuların her birine 100 mcL FCM Tampon ve kısım 20 mcL boncuk tekrar </li><li> Örnekleri florokrom-konjuge mAbs ekleyin. FCM üreticinin talimatlarına göre veya ampirik olarak tespit yoğunluklarına göre leke gerçekleştirin. Tüp başına ya da iyi bir başlangıç ​​noktası olarak, hisse senedi antikor çözüm 0,2-1 mcL (0,2-1,0 mg / ml) ekleyin ve 40 dakika buz üzerinde inkübe edin. </li><li> Yıkama 1,0 mL buz FCM Tampon 4 20.000 g de santrifüj 1.5 ml tüpler iki kez boncuk probed ° C her yıkamadan sonra 3 dakika. Yavaşça, boncuklar rahatsız etmemek için özen, her yıkamadan sonra süpernatantı çıkarın. Baca alt plaka, 4, 5 dakika boyunca 1000g santrifüj, 0.2 ml buz FCM Tampon ile iki kez yıkamak ° C Her yıkamadan sonra. Bir çok kanallı pipet bu adım için yararlıdır. Bir baca alt plakası boncuk süpernatant kaldırmak için, plaka &#39;hareketiyle bir kez, sonra, hala baş aşağı iken, kuyuların kenarlarında herhangi bir damlar kurulayın. Her kuyuda rezidüel hacim, boncuklar içeren yaklaşık 20 mcL. </li><li> Numune başına 200 mcL FCM Tampon boncuklar tekrar Etiketli FACS tüpler aktarın. Örnekleri FCM hazır. </li></ol> 4. FCM Acquisition Bu protokol açıklanan KML boncuklar, çapı 3 ila 5 mikron, suskun bir fare lenfosit çapı yaklaşık yarısı. Bu nedenle, bu ölçekte kayıt boncuk olayların nüfus için elle Forward Scatter (FSC) amfi kazanç ve Side Scatter (SSC) gerilimi artırmak için gerekli olabilir. Bireysel IP boncuk tek bir sıkı kümelenmiş nüfus oluşturmalıdır. Ayarları ve kapı boncuk çiftlerin ve enkaz dışlamak için ayarlanabilir olmalıdır. <ol><li> Önce çalışan boncuk boncuk standartları, bazı sitometrelerinde numune girişi çevreleyen damlacık çevreleme kol çıkarın. Bu giriş temizlemek ve başka bir örnek üzerinden yürütülmektedir boncuk önleyecek şekilde kılıf sıvı örnekleri arasında damla izin verin. </li><li> Negatif ve pozitif floresan aşırı log ölçekli x ekseni aynı anda görünecek şekilde ayarlarını etiketsiz boncuk, floresan bir negatif kontrol ve Rainbow Kalibrasyon Parçacıklar (RCPs), floresan bir pozitif kontrol, kullanın. Bu deneysel örneklerinden floresan ölçekli olacağını garanti eder. Böylece FSC ve SSC parametrelerin yanı sıra kapı konumu, geçici olarak bu örnek için değişiklik gerekebilir RCPs KML boncuk daha küçük olduğunu unutmayın. </li><li> RCPs ile kalibrasyon tamamlandıktan sonra etiketsiz-boncuk ayarlarına geri dön. Örnek başına sadece bir floresan prob kullanılması durumunda, hiçbir floresan tazminat gereklidir. Genel olarak, tek başına bir FCM örnek florokrom-konjuge mAb ile multiprotein kompleksleri probu, ve çeşitli alt birimden özel mAbs paralel boncuk örnekleri boyama ile birden fazla etkileşim ortakları incelemek. </li><li> Floresan veri dosyaları Edinme ve kaydedin. Analizi sonra flow sitometri gibi CellQuest, FlowJo yazılım veya CFlow kullanılarak yapılabilir. </li></ol> 5. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2066/2066fig1.jpg" alt="Şekil 1" /> Şekil 1. Fare suşu TCR/CD3 multiprotein kompleksi için IP-FCM T hücreleri BALB / c 1% digitonin parçalanan, ve anti-CD3ε boncuklar kullanarak IP-FCM lizat maruz kaldı. Yakalanan kompleksleri TCR-β önemli miktarda (mor bölge) bulunan ve co-ilişkili CD3-ε (yeşil), ancak yakın gibi Thy1.2 gibi diğer proteinlerin arka plan düzeyleri (alakasız immünglobulin probu, pembe iz tarafından tanımlanan), CD45, ya da H-2K (d) (kahverengi, turuncu ve mavi izleri, sırasıyla). Daha önce yayınlanmış benzer sonuçlar: 1-6.

<ol>
	<li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+detection+and+quantitative+analysis+of+native+protein-protein+interactions+and+multiprotein+complexes+by+flow+cytometry.">High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry.</a> Sci STKE. , pl2-pl2 (2007).</li><li>Schrum, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+multiprotein+complexes+by+flow+cytometry.">Visualization of multiprotein complexes by flow cytometry.</a> Curr Protoc Immunol. Chapter 5, 9-9 (2009).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+division+and+death+are+segregated+by+mutation+of+TCRbeta+chain+constant+domains.">T cell division and death are segregated by mutation of TCRbeta chain constant domains.</a> Immunity. 21, 515-526 (2004).</li><li>Teixeiro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+T+Cell+receptor+signals+determine+CD8++memory+versus+effector+development.">Different T Cell receptor signals determine CD8+ memory versus effector development.</a> Science. 323, 502-505 (2009).</li><li>Treves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctate+is+a+key+element+in+calcium+entry+induced+by+activation+of+InsP3+receptors+and/or+calcium+store+depletion.">Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion.</a> J Cell Biol. 166, 537-548 (2004).</li><li>Gil, D., Schrum, A. G., Alarcon, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+engagement+by+peptide-MHC+ligands+induces+a+conformational+change+in+the+CD3+complex+of+thymocytes.">T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.</a> J Exp Med. 201, 517-522 (2005).</li></ol>

Protein-protein etkileşimleri hakkında bilgiler, sinyal iletimi, soy olgunlaşma, hücre döngüsü ilerlemesi, ve apoptozis kaskadlar gibi birçok hücresel süreçlerin analiz son derece alakalı. IP FCM proteinlerin etkileşimi incelemek ve kendi ana yapısındaki multiprotein kompleksleri üyeleri tanımlamak için, hızlı, kantitatif ve hassas bir şekilde sağlar. Boncuk ve hücre lizatları ile inkübe birleştiğinde, bir günde ve probed ve ertesi gün analiz edilebilir olabilir. 96-plaka biçimi, etkin veri toplama, istatistik veya tarama amacıyla sağlamak için bir anda analiz edilmesi gereken çok sayıda numune sağlar. Floresan boncuk standartlarını kullanarak, her bir boncuk tarafından yakalanan proteinlerin sayısını tahmin edilebilir. Çok küçük kaynak materyal yakalama ve tespiti için gerekli, bu yüzden sınırlı örnekler ve kıt analitler hala çok etkileşim için analiz edilebilir. IP-FCM fizyolojik olmayan bir ortamda proteinlerin karıştırma, genetik mühendisliği, epitop etiketleme, denatürasyon, ya da in-vitro gerektirmese de, uygulanabilirliği ile erişilebilir ve değerli bir araç, bu ve diğer teknikler ile birleştiğinde olabilir birçok biyolojik sistemler. Sorun Giderme: Birçok IP FCM deneyler yararlı protein etkileşim verileri ilk kez denenir üretir. Ancak, IP-FCM optimizasyonu, boncuk, protein kompleksi yakalama ve bağlayıcı floresan prob antikor konjugasyon artırabilir. IP antikor konjugasyon verimliliği doğrudan bir anti-immünglobulin antikor ile birleştiğinde boncuk problama tarafından tespit edilebilir. Bu verimliliği düşükse, kaplin reaksiyon sırasında antikor konsantrasyonunu arttırarak daha fazla IP antikorları her boncuk takmak için izin verebilirsiniz. Bu analitlerin gelişmiş yakalama ve algılama, IP boncuk toplu bağlama kapasitesi artırabilir. Diğer birincil amin içeren moleküllere (örneğin Tris, sığır serum albumin), bu düşük mAb kaplin boncuk eki ve sonuç için mAb ile rekabet gibi kaplin reaksiyon sırasında mevcut olmamalıdır. Boncuk mAb konjugasyon başka nedenlerden dolayı sorun olursa, boncuk yerine avidin / streptavidin birleştiğinde olabilir ve biotinlenmiş antikorlar daha sonra non-kovalent bağlı immunprecipitation için kullanılan olabilir. Iyi antikor konjugasyon rağmen, boncuk ilişkili floresan ilk algılama düşük olabilir. Birincisi, karmaşık yakalama kendisi düşük olabilir. IP-FCM, birim lizis hacim başına parçalanan hücrelerin sayısı artan analit konsantrasyonuna bağlı olduğundan analit yakalama ve algılama artırabilir. Ayrıca, yakalama, yakalanan az boncuklar arasında kompleksleri, ve probed boncuk başına ortalama floresan sonuçları dağıtır lizat ile inkübe IP boncuk sayısının azaltılması artabilir. İkincisi, yakalanan kompleksleri prob antikor erişim sterik immunoprecipitating antikor tarafından engellendiği mümkündür. Bu durumda, sorun ve / veya prob yakalamak için farklı antikorları kullanarak ele alınabilir.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free)</td>
<td>Reagent</td>
<td>Interfacial Dynamics Corporation/Molecular Probes</td>
<td>2-5000</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>Rainbow Calibration Particles</td>
<td>Reagent</td>
<td>Spherotech, Inc.</td>
<td>RCP-30-5A</td>
<td>Store at 4&deg;C.</td>
</tr>
<tr>
<td>2-[N-Morpholino] ethanesulfonic acid (MES)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-5287</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC)</td>
<td>Reagent</td>
<td>Pierce Sigma</td>
<td>22980 E-6383</td>
<td>Store at -20&deg;C under desiccating conditions.</td>
</tr>
<tr>
<td>Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-2714</td>
<td> Prepare 100x stock solution in H2O. Aliquot and store at -20&deg;C. Handle with gloves.</td>
</tr>
<tr>
<td>PCR plates, 96-well 0.2mL, natural, tall chimney</td>
<td>Reagent</td>
<td>Fisher</td>
<td>08-408-230</td>
<td>For use in place of microcentrifuge tubes during FCM staining</td>
</tr>		</tbody>
		</table>
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ip-fcm:  immunoprecipitation detected by flow cytometry

Flow sitometri (IP-FCM) tarafından tespit Immunoprecipitation protein-protein etkileşimleri tespit edilmesi ve ölçülmesi için etkili bir yöntem. Temel ilke yakalanan birincil analit multiprotein kompleksleri içinde fiziksel olarak ilişkili diğer molekülleri ile birlikte tespit edilebilir neyin sandviç ELISA uzanır. Prosedürü, monoklonal antikorlar (mAb) belirli bir protein ilgi immunoprecipitating hücre lizatları bu boncuklar kuluçka florokrom-konjuge problar ile yakalanan protein kompleksleri sondalama ve boncuk ilişkili floresan flow sitometri analiz ile polistiren lateks mikro kovalent kaplin içerir. IP-FCM, son derece duyarlı, kendi ana (denatüre) devlet proteinlerin analizi sağlar ve iki yarı-kantitatif veya kantitatif analiz için uygun. IP-FCM ek avantajlar olarak, bir akış sitometresinde dışında hiçbir genetik mühendisliği ya da özel ekipman gerektirir ve yüksek hacimli uygulamalar için kolayca adapte edilebilir.

Watch this Scientific Journal Video about IP-FCM:  Immunoprecipitation Detected by Flow Cytometry at JoVE.com

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Genetik mühendisliği ya da büyük örneklem büyüklüğü gerektirmeden, hassas bir doğal protein-protein etkileşimleri, sağlam, biyokimyasal değerlendirme sağlar IP-FCM yöntem sunulmuştur.

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 ...

ip-fcm-immunoprecipitation-detected-by-flow-cytometry

Research

JoVE Journal

Biology

17.4K Görüntüleme.  Mayo Clinic. Genetik mühendisliği ya da büyük örneklem büyüklüğü gerektirmeden, hassas bir doğal protein-protein etkileşimleri, sağlam, biyokimyasal değerlendirme sağlar IP-FCM yöntem sunulmuştur.

Video: IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

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 ...

Biyoloji

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 ...

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 ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

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 ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Akışı - Akım Sitometrisi ile optogenetics birleştiren

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication izole yumru önceki Akış Sitometresi tarafından ölçme

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Dictyostelium discoideum Macropinocytosis akış sitometresi tarafından yüksek üretilen iş ölçümü

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.