Worcester Politeknik Enstitüsü Bölümü Kimya ve Biyokimya finansman için teşekkür etmek istiyorum.

1. Hücre Hatları, Transfeksiyon Transfeksiyon Yöntem 1 (Lonza, artan primer hücre transfeksiyon verimlilik için Nucleofection) <ol><li> Hücreler son geçiş ve önce transfeksiyon log fazı büyüme olmalıdır. Fosfat içinde hücreler yıkayın trypsinization tamponlu salin (PBS) ve hasat hücreleri. </li><li> 1500 xg&#39;de 5 dakika santrifüj hücreleri toplayın. </li><li> 6 plaka sterilize kapak fişleri ekleyin. Kapak fişleri gelişmiş hücre adezyon kaplı olabilir. Yeri 1.5 ml kültür ortamı (bu durumda Dulbecco&#39;nun modifiye Eagle orta (DMEM),% 10 fetal sığır serumu (FBS)) ve her kuyuya inkübatör ortam dengelenmesi. </li><li> Fosfat minimal volüm trypsinization ve tekrar süspansiyon hücreleri tarafından Hasat tamponlu salin (PBS). </li><li> Hücre sayımı ve tabanda örnek başına ~ 5x10 5 hücre sağlamak için doğru miktarda santrifüj. </li><li> Numune başına 100 mcL oda sıcaklığında Nucleofector çözüm hücreleri dikkatli bir şekilde tekrar </li><li> 5 mg plazmid DNA ile birleştirin ve hücre süspansiyonu 100 mcL Nucleofector Küvet hava kabarcıkları dahil etmek için dikkatli bir örnek aktarmak. </li><li> Uygun Nucleofector Programı (minimal mortalite ile optimal transfeksiyon etkinliğini kurmak için önceki testi gerekebilir) seçin. </li><li> Nucleofector Küvet Tutucu hücre / DNA süspansiyon içeren küvet takın ve seçilen program başlar. </li><li> Tamamlanmasının ardından, küvet kaldırmak ve (6 plaka alınan) ~ 500 mcL ön dengelenmiş kültür ortamı ekleyin. </li><li> Hemen yanı başına 1.5 mL son hacmi ile 6-plaka içine örnek aktarmak. Hücreler kontrolleri için, karma nüfuslu ya da ayrı ayrı ya kaplama olmalıdır. </li></ol> Transfeksiyon Yöntemi 2 (Qiagen Effectene transfeksiyon hücre hatları için) <ol><li> Her plazmid DNA örneği 0.8 mg 200 mcL EC tampon ekleyerek ilk Qiagen Effectene Reaktif transfeksiyon kompleksleri kullanarak hazırlayın. </li><li> Her numune için 6.4 mcL arttırıcı reaktif, tüp Flicking kısaca karıştırın ve oda sıcaklığında 2 dakika inkübe edin. </li><li> Tüp Flicking tarafından her örnek, mix 20 mcL Effectene reaktif ekleyin ve oda sıcaklığında 15 dakika inkübe edin. </li><li> Bu kuluçka sırasında, transfeksiyon için ilgi iki hücre hatları hazırlamak. Son geçişli hücreler (&lt;% 80 izdiham) yıkayın kez fosfat tamponlu salin (PBS). 1.5 ml taze ısıtılmış ve dengelenir büyüme orta Ekle (Bu durumda Dulbecco&#39;nun modifiye Eagle orta (DMEM),% 10 fetal sığır serumu (FBS)). </li><li> 15 dakika boyunca inkübe transfeksiyon kompleksleri sonra, her bir örnek 1.2 mL medya aktarın. Pipetleme, ve 6-plaka hemen her iki kuyu için yaklaşık 700 mcL kompleksleri transferi ile karıştırın. Damla damla ekleyin ve plaka dönen hafifçe karıştırın. Hücrelerin en az 3 saat transfect için izin ver (hücre tipi ile değişiklik gösterebilir). </li><li> Sterilize kapak fişleri yeni bir 6-plaka ekleyin. Kapak fişleri gelişmiş hücre adezyon kaplı olabilir. </li><li> Hücrelerin transfekte sonra, her bir kuyunun yaklaşık 100 mcL tripsin çözüm ekleyerek, kısaca her bir kuyunun tam kat plaka ajitasyon ve hemen tripsin aspire PBS ile iki kez hücreleri yıkayın ve minimal trysinization hücreleri tarafından hasat. Hücreleri kaldırdı sonra, 1.5 mL taze medya ekleyin. </li><li> Bu noktada, hücrelerin steril bir örtü fişleri üzerine karışık veya ayrı olarak nüfus (kontrol) kaplama olmalıdır. </li><li> Hücreler 12 saat kurtarmak için izin verin. </li></ol> 2. Hücre Fusion <ol><li> Hücrelerin kültür ortamları çıkarın ve PBS ile iki kez yıkayın. </li><li> Bu noktada, protein sentezini inhibe sikloheksimid (100 mg / ml) ile hücrelerin tedavisi için tavsiye edilir. Hücreler 2 saat süreyle inkübe ve sonra hücreler PBS ile iki kez yıkamak için izin verin. </li><li> 125 saniye boyunca oda sıcaklığında serum serbest DMEM% 50 polietilen glikol 1000 (PEG 1000) bir çözüm onları teşhir ederek Sigorta hücreleri. </li><li> PEG 1000 çözüm kaldırmak ve 2 ml taze ısıtılmış ve dengelenir medya eklemek hücreleri PBS ile yıkayın. </li><li> Hücreleri için 18 - 24 saat inkübe için izin verin. </li></ol> Floresan Mikroskopi <ol><li> Kültür ortamı çıkarın ve hücreler PBS içinde iki kez yıkayın. </li><li> Her iyi bir% 4 paraformaldehid solüsyonu (PBS) 1 ml ekleyerek hücreleri saptamak. 15 dakika boyunca yavaşça ajitasyon. </li><li> Sabit hücreler PBS içinde iki kez yıkayın. </li><li> Plaka kapağını dikkatlice fişleri kaldırmak, aşırı PBS kapalı fitil. Üzerine mikroskop yüklenen montaj orta (% 90 gliserol, 100 mM Tris, pH 7.5,% 2 DABCO (1,4-diazabicoyclo oktanlı) içeren DAPI (4 &#39;,6-diamidino-2-phenylindole) ile bir damla slaytlar. Haricindekileri </li><li> Slaytları fazla montaj orta çıkarın ve mühür cooje ile ver sığar. </li><li> Konfokal veya Epifloresans mikroskobu ile hücre gözünüzde canlandırın. </li></ol> 3. Temsilcisi Sonuçlar Şekil 2 birincil fibroblastlar ve H1299 küçük hücreli dışı akciğer kanseri hücreleri kullanarak bir örnek heterokaryon deney gösterir. Bu deneyde ilgi protein (Tavuk Anemisi Virüs VP3) Epifloresans mikroskopi tarafından görüntülenmiştir olarak öncelikle birincil hücre tipleri sitoplazmik yerelleştirme ve transforme hücre tipleri nükleer lokalizasyon gösterir. Protein H1299 hücrelerinde birincil sünnet derisi fibroblastlar (PFF) veya dsRed GFP ya da bir füzyon (pEGFP N1 vektör, Clontech) (dsRed-N1 vektörü, Clontech) olarak ifade edilir. Kendi füzyonlar içeren kontrol numuneleri (üst iki satır), kararlı durum lokalizasyon desen hiçbir değişiklik multinükleer hücreler gösterir. Bu tür değişiklikler aksi füzyon koşulları veya syncitia oluşumu hassasiyeti nedeniyle meydana gelebilir. Heterokaryon füzyonlar (alt sıra) nükleer GFP-erimiş birincil hücre kaynaklı protein ve transforme hücre malzeme giriş yanıt dsRed erimiş protein ile ko giriş göstermektedir. Gösterilen örnek, hem Fluoroprotein sinyallerin varlığını gösterdiği gibi, sadece iki hücre, her tür, oldukça nadir görülen bir heterokaryon füzyon. Nükleositoplazmik mekik faaliyet yerelleştirme geçişler bu tür tespit ek olarak, hem de çekirdeklerin tek bir fluorofor varlığı kolayca tespit edilebilir. Bu tip tayini 01:01 hücre füzyonlar incelenmesi ve çeviri inhibisyonuna bağlı olacaktır açıktır. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Şekil 1" /> Şekil 1. Heterokaryon yöntemin birincil fibroblastlar ve H1299 küçük hücreli dışı karsinom hücreleri kullanarak akış şeması ilgi gen iki floresan protein genleri tek bir kare füzyon üretmek için klonlanmış. Daha sonra hücre hatları geçici ya da inşa transfekte ve kurtarmak için izin vardır. Heterokaryon oluşumu daha fazla inkübasyon polietilen glikol (PEG) ile kısa süreli tedavi ile meydana gelir. Son olarak, faiz protein alt hücresel yerleşimi floresan mikroskobu çeşitli formları kullanarak görülmektedir. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Şekil 2" /> Şekil 2. Nükleositoplazmik kaçakçılığı ve Tavuk Anemisi Virüs VP3 proteinin aktivitesi mekik heterokaryon füzyon tarafından görüntülendi. Üst satır: dsRed-VP3 ifade kendinden erimiş H1299 hücreleri Temsilcisi Orta sıra: PEG füzyon sonra GFP-VP3 ifade birincil sünnet derisi fibroblast hücreleri (PFF) Temsilcisi Epifloresans görüntüleri Alt satır: PFF ve H1299 hücreleri Heterokaryon füzyon. Sarı GFP ve dsRed sinyaller ko gösterir. Hücreler bir Leica AF6000E floresan mikroskop ve Leica görüntüleme yazılımı kullanılarak görüntülenmiştir.

<ol>
	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

Burada sunulan heterokaryon protokol hücre tipine spesifik lokalizasyon davranış yanı sıra Nükleositoplazmik mekik faaliyet karakterizasyonu için etkin ve kolay yorumlanabilir bir yöntem sağlar. Bu yöntem, floresan analizi duyarlılık yanı sıra yetenek görüntü canlı hücreleri ve protein dinamiği çalışmaları gerçekleştirmek yararlanır. Tekniği, pek çok farklı hücre tipleri için kolayca adapte ve hem canlı ve sabit hücre görüntüleme için uygun. Örneğin, ters floresan mikroskopi canlı görüntüleme ve zaman atlamalı video yakalama için kullanılabilir. Buna karşılık, kararlı durum lokalizasyonu veya ayrık lokalizasyonları daha ayrıntılı konfokal görüntüleme belirlenmesi için iki Epifloresans mikroskopi monte hücreleri kullanılıyor olabilir. Ayrıca, bu tür photobleaching sonra floresan kurtarma (sıkı bağlamak), ya da floresan photobleaching içinde kaybı (FLIP) gibi teknikler, lokalizasyon kinetiği 1 tespitinde kullanılabilir . Protokol alternatif fluorophores birleşme 2 konser yapılacak floresan rezonans enerji transferi (FRET) gibi protein etkileşim deneyler için izin verecek. Leptomycin B veya olumsuz egemen olarak hücresel ticareti Çeşitli inhibitörleri Ran GTPaz yapıları, özellikle ithalat veya ihracat yollar 5,6,9 bağımlılığı kurmak için kullanılabilir . <ul><li> Faaliyet mekik içeren bazı deneylerde, bakım, yeni protein sentezi nedeniyle yerelleştirme etkilerinden kaçınmak için gerekli önlemler alınmalıdır. Bu durumlarda, çeviri inhibitörleri veya sınırlı bir süre noktaları kullanılmalıdır. Ancak, bir olasılık translasyonel inhibisyonu nedeniyle eserler kalır. Biri Hücre füzyonlar her hücre tipi içeren, mekik ve yerelleştirme davranışı net yorumlanması için en çok istenen. Hücre sayısı ve füzyon koşulları (zamanlama ve konsantrasyon) optimizasyonu tür olumlu 01:01 füzyon olayları teşvik etmek için gerekli olabilir. </li><li> Protokol bazı adımlar bu optimizasyon kullanılan özel hücre tipleri bağlı olarak gerekli olacaktır dikkat etmek önemlidir. Füzyon etkinlik, Transfeksiyonu verimlilik ve füzyon sonra canlılığını deneysel işlemin Yönleri hücre tipleri arasında büyük farklılık gösterebilir. Özellikle, birincil hücre tiplerinin karmaşık kültür koşulları ve düşük transfeksiyon verimliliği nedeniyle yönetmek için zor olabilir. Lonza Nucleofector cihazı gibi teknolojileri kullanımı hücrelerin hızlı bir şekilde işleme yanı sıra önemli ölçüde artmıştır transfeksiyon verimliliği izin verebilirsiniz. Ayrıca, elektroporasyon yöntemleri takip füzyon adımları askıya hücre kolay transferi için izin verir. </li></ul>

<table><tbody><tr><td>Effectene reagent</td><td>Qiagen</td><td>301425</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>Trypsin</td><td>Fisher Scientific</td><td>SH3023602</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Fisher Scientific</td><td>SH30243FS</td><td>High glucose</td></tr><tr><td>paraformaldehyde</td><td>Fisher Scientific</td><td>O4042-500</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542-10MG</td><td></td></tr><tr><td>Hyclone Serum (fetal bovine)</td><td>Thermo Fisher Scientific, Inc.</td><td>SH3008803HI</td><td></td></tr><tr><td>Falcon 6-well plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Polyethylene glycol 1000</td><td>Fisher Scientific</td><td>528875-25GM</td><td></td></tr><tr><td>Cover slips</td><td>Fisher Scientific</td><td>12-545-85</td><td></td></tr><tr><td>DABCO</td><td>Sigma-Aldrich</td><td>D2522-25G</td><td></td></tr><tr><td>Nucleofector HDF kit</td><td>Lonza Inc.</td><td>VPD-1001</td><td></td></tr></tbody></table>

heterokaryon technique for analysis of cell type-specific localization

Önemli bir sayıda proteinler, hücre içi ticareti veya nucleocytolasmic mekik tarafından düzenlenir. Bu proteinler, RNA ve protein nükleer ithalat / ihracat, transkripsiyonal yönetmelik, ve apoptosis gibi hücresel fonksiyonları çeşitli bir dizi gösterir. İlginçtir, hücre bölünmesi, farklılaşma ve dönüşüm de dahil olmak üzere önemli hücresel yeniden yapılanma, sık sık bu tür faaliyetler 3,4,8,10 içerir. Bu proteinlerin ve kendi düzenleyici mekanizmaların detaylı bir çalışma bu lokalizasyon değişiklikleri zor olabilir uyarılması ve kendilerini izlemek için oldukça dinamik ve zor olabilir hareketleri gibi zor olabilir. Hücresel karsinojeneze içeren çalışmalar, örneğin, anlayış yollar ve birincil, normal hücreleri ve transforme hücreleri 6,7,11,12 arasında farklılık protein aktiviteleri yararlanmaya devam. Birçok protein değişmiş dönüşümü sırasında veya lokalizasyonu, dönüşümün bir sonucu olarak verimli bir şekilde bu proteinlerin karakterize etmek için yöntemler ve ilaç hedef karsinojeneze ve açık yeni alanlar anlayışı geliştirmek stand katıldığı yollar gösterir. Burada protein ticareti analizi için bir yöntem mevcut ve birincil ve transforme memeli hücreleri arasındaki aktivite mekik. Bu yöntem, kararlı durum ya da dinamik protein lokalizasyonu tespit etmek için kullanılan esnek bir protokol sağlamak için floresan mikroskobu ile heterokaryon füzyonlar nesil birleştirir. Şekil 1&#39;de gösterildiği gibi, iki ayrı hücre tipleri geçici ilgi gen bağlı bir Fluoroprotein geni taşıyan plazmid yapıları ile transfekte. Ifade sonra, hücreler polietilen glikol kullanarak erimiş ve protein lokalizasyonu daha sonra çeşitli yöntemler kullanarak görüntülü olabilir. Burada sunulan protokol, özel teknikler ilave edilebilir temel bir yaklaşımdır.

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

Mekik hücre tipine spesifik protein yerelleştirme ve Nükleositoplazmik karakterizasyonu için esnek ve verimli bir yöntem açıklanmıştır. Bu heterokaryon yaklaşım görüntü protein lokalizasyonu hücre füzyonu sonra floresan etiketli füzyon proteinleri kullanır. Protokol kararlı durum lokalizasyonu veya canlı hücre görüntüleme dayalı daha dinamik tespitler mükellef.

Hücre Tipi özel Yerelleştirme Analizi için Heterokaryon Tekniği

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

Research

JoVE Journal

Biology

16.4K Görüntüleme.  Worcester Polytechnic Institute- WPI. Mekik hücre tipine spesifik protein yerelleştirme ve Nükleositoplazmik karakterizasyonu için esnek ve verimli bir yöntem açıklanmıştır. Bu heterokaryon yaklaşım görüntü protein lokalizasyonu hücre füzyonu sonra floresan etiketli füzyon proteinleri kullanır. Protokol kararlı durum lokalizasyonu veya canlı hücre görüntüleme dayalı daha dinamik tespitler mükellef.

Video: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Bir moleküler karakterize etmek için Kombinatorik tek hücreli yaklaşım ve insan kök ve yaratıcı nüfus Immunophenotypic heterojen

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Genetik

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Çift nokta MARCM kullanılması Hücre Lineage Analizleri ve Gen Fonksiyonu Çalışmaları

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Gelişim Biyolojisi

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Kromozom segregasyon mitoz sırasında canlı hücre görüntüleme

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Biyoloji

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Sıçan beyin dokusunda incelemek için THP kullanılarak hücre-tipi-spesifik gen ifadesi

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Nörobilim

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Retinal Ganglion Hücrelerinin Tanımlanmış Alt Kümelerinin Tek Hücreli RNA-Seqi

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D Nükleer teşkilatında değişiklikler Eğitim için 3D-korunmuş Tane Çekirdekle ilgili Kombine İmmünofloresan ve DNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Dan Tagged Çekirdeğin Affinity-tabanlı yalıtım<em&gt; Drosophila</emGen ekspresyon analizi için&gt; Dokular

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

Çok renkli yerelleştirme mikroskobu canlı memeli hücre organelleri tek membran proteinlerin

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Source: Aishwarya, R., et al. <a href="https://app.jove.com/v/64085">Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy.</a> J. Vis. Exp. (2022).
This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron microscopy.

Proteinlerin hücre altı lokalizasyonunu görselleştirmek için bir immünoetiketleme tekniği

1. Animal preparation

	Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen ...