Bu çalışma, Ulusal Bilim Vakfı (hibe yok. DBI 0.519.984) ve Ulusal Sağlık Enstitüleri (hibe yok. 5R01GM078279) tarafından desteklenen ..

1) bitki materyali hazırlanması <ol><li> Protoplast sindiren enzimleri hücre duvarının sağ karışımı 1 olduğunu sağlanan pek çok farklı bitki türlerinin ve dokular elde edilebilir. Tam ölçekli bir deney yapılmaktadır önce, küçük ölçekli bir sindirim malzeme, doku protoplasting verimliliği, enzimler, vb değerlendirmek ve hücre sıralama için yüzde pozitif hücrelerin tahmin etmek için tavsiye edilir. Burada, hücre tipi, özellikle açık yeşil flüoresan protein (GFP) kullanılır olduğunu Arabidopsis thaliana fide kökleri elde edilen protoplast . Endodermis ve latent merkezi P SCR tarafından işaretlenir: GFP ve P WOX5 latent merkezi: GFP 2,3 (Şekil 1). </li><li> Kökleri (% 0.22 w / v Murashige ve Skoog Bazal Orta [Sigma],% 1 büyüme ortamına yoluyla büyümeye olanak sağlar; naylon filtre (NITEX 250 mikron mesh); Fideleri (Şekil 2a Sigma) phytatrays hydroponically yetiştirilen w / v sakaroz,% 0.05 w / v MES [2 - (N-morfolino) ethanesulfonic asit], KOH ile pH 5.7). Alternatif olarak üst üste dikey olarak yerleştirilmiş% 1 agar plaklarına bir naylon filtre (100 mikron mesh) (Şekil 2b), bitkiler yetiştirilebilir. </li><li> Köklerin hasat sadece yardımların yukarıda belirtilen filtreleri kullanmak istenirse, o da kolay, fide ek tedavi kolaylaştırır. Filtreler fide ilgi bir katalizör ile desteklenmiş yeni phytatrays veya agar plaklarına topluca aktarılacak izin verir. Örneğin, phytatray seti Arabidopsis fide azot tedavisi 4 transkripsiyonel yanıt hücre tipine özgü analizi için kullanılan olmuştur. </li><li> Mikroskobik floresan işaretleyici doğru ifade olduğunu (kendilerini tedavi etkilemiş olabilir, hücre tipi özel işaretleri olarak tedavi seçeneği kullanarak özellikle) emin olun. Bu durumda, fide, floresan mikroskop altında incelenmelidir (Şekil 1 Nikon). Hücre tipi özel floresan işaretleyici hatları tam olarak hangi hücre tipleri işaretlenmiştir belirlemek ve ifade değişkenlik belirlemek için bir konfokal mikroskop altında başlangıçta karakterize gerektiğini unutmayın. </li></ol> 2) Hazırlık protoplasting çözüm <ol><li> 1.25% w / v selülaz (Yakult),% 0.3 w / v Macerozyme (Yakult), 0.4 M D-mannitol, 20 mM MES ve 20 mM KCl demineralize su (1 M stok) çözülür ve pH 5.7 &#39;ye ayarlayın 1 M Tris / HCl pH: 7.5. Bu çözüm biraz bulanık olacaktır. </li><li> Çözüm ile 55 Isı ° C, 10 dakika (çözüm açık dönecek) ve% 0.1 eklemeden önce oda sıcaklığına kadar soğumasını bekleyin w / v BSA (sığır serum albumin), 10 mM CaCl 2 ve 5 mM β- mercaptoethanol . </li></ol> 3) Hasat ve bitki materyali, protoplasting <ol><li> Kökleri bir neşter ile naylon mesh onları kazıyarak hasat ve protoplasting çözüm içeren bir balona yatırılır. Genel olarak, 10 ml protoplasting çözüm 1.500 fide kökleri başına kullanılır. </li><li> Bir saat boyunca oda sıcaklığında matara (75 rpm) hafifçe çalkalayınız. Daha uzun bir kuluçka süresi protoplast verimi artırabilir, ama aynı zamanda gen ekspresyonu üzerine kendisi protoplasting etkisi eklemek olacaktır. </li><li> 40 mikron hücre süzgeç (BD Falcon) protoplast çözüm Filtre ve konik 15 ml tüpler (BD Falcon) üzerinden çözüm bölmek. </li><li> 500 G. Not Bu santrifüj hızı kullanılan protoplast tipi, kırılganlık ve enzimatik tedavi sırasında üretilen hücre enkaz miktarı bağlı olacağını, 10 dakika boyunca bir salıncak-kova santrifüj tüpleri dönerler. </li><li> Süpernatantı çıkarın, kalan çözüm protoplast tekrar süspansiyon ve mikroskobik olmadığını kontrol edin (Şekil 3). </li><li> Protoplast sayısını ve yoğunluğunu tahmin etmek için bir hemasitometre yararlanın. Hücre yoğunluğu örnek enjeksiyon hızı, FACS hücreler sıralamak için gerekli olan ikinci sınıf başına olaylar ve bu nedenle toplam süre (bkz. bölüm 4.3) belirleyecektir. </li><li> 3.7) FACS doğrudan devam etmek ya da olmadan, W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM (MES), KOH ile pH 5.7 &#39;ye ayarlamak) veya protoplasting çözüm olarak bir kuluçka çözüm, protoplast yıkayın ve tekrar süspansiyon Ya enzimler eklendi. Özellikle transkripsiyonel değişiklikler bakarak, protoplast Bu nedenle protoplasting çözüm protoplast tutmak ve devam etmek için tavsiye edilir tutulur tampon değiştirme gibi örneklerinin gen ekspresyonu etkileyebilecek koşullara maruz kalma, en aza indirmek için önem taşımaktadır. mümkün olan en kısa sürede FACS. </li></ol> 4) Floresan protoplast Aktif Hücre Ayırma <ol><li> Açın ve hücre sıralayıcı hazırlamak. Burada, bir FACSAria (BD) kullanılır. </li><li> 100 &amp; m olan bir akım akışıu; m meme ve 20 psi kılıf basınç. </li><li> Hücre yoğunluğu ve numune enjeksiyon hızı, mümkün olan en iyi verim veya hızlı ulaşılabilir hızı istenilen olup olmadığına göre belirli bir deneme için ayarlanabilir. Biz 10.000.000 hücre / ml &#39;ye kadar yoğunlukları ile başarılı bir şekilde sıralanır var. </li><li> Protoplast sedimantasyon önlemek için FACS örnek ajitasyon seçeneği kullanın. 1: FACS tıkanma bir sorun ise, üç olası sorun giderme adımları vardır. Bir örnek-line Backflush gerçekleştirin. 2. Yoğunluğu azaltmak için protoplast süspansiyon sulandırınız. 3. Protoplast çözümü, santrifüj ve tabanda sonra filtrasyon adım (3.3) tekrar temizleyin. </li><li> 488 nm lazer GFP ve 610/20 nm eksitasyon sonra kızıl spektrumu otofloresansı (RSA) 530/30 nm&#39;de forward scatter (FSC), yan dağılım (SSC) ve emisyon ölçümü için cihazın hazırlayın. Bunlar özünde GFP pozitif protoplast izole etmek için kullanılan parametrelerdir. Burada voltaj ayarları aşağıdaki gibidir: FSC - 60V, SSC 250V, GFP 350V ve RSA 335V. Not optimum voltaj ayarlarını her FACS için farklı olacaktır ve hatta hücre sıralayıcı ömür boyu ayarlanabilir gerekecektir. </li><li> Forward scatter karşı tarafında dağılım için dotplot kurarak başlayın. Ölçülen olayları komplo merkezli böylece voltaj ayarları uygulayın. </li><li> Sonra, karşı kırmızı yeşil floresan sinyallerin bir nokta arsa oluşturun. Wild-tip (non-GFP) protoplast süspansiyon bakarken ölçülen olayları komplo merkezli çapraz bir nüfus verim böylece voltaj ayarları uygulayın. GFP işaret çizgisine türetilmiş bir protoplast süspansiyon, wild-tip örnekleri görülen yeşil floresan olaylar asla net bir nüfus üretecektir. </li><li> GFP ve RSA arasındaki spektral örtüşme ayarlamak için tazminat kısıtlamaları ayarlayın. Uygun tazminat kısıtlama ayarlarını olmayan GFP protoplast ve enkaz GFP pozitif protoplast daha iyi ayrılması için izin verecektir. RSA, eksi 17,91% GFP: Burada kullanılan kısıtlamaları aşağıdaki gibidir. </li><li> Non-GFP protoplast bir negatif kontrol kapısı sınırları (Şekil 4) tanımlanmasında yardımcı olmak için kullanılması gereken GFP pozitif olayları tanımlamak için bir kapı ayarlayın. </li><li> Analiz küçük enkaz bırakmak için ileri bir dağılım kesme uygulayın. FSC vs SSC arsa GFP pozitif olaylar kesme yerleştirme belirlemenize yardımcı olmak için gözünüzde canlandırın. Burada, kesme 5.000 olarak belirlendi. FACS enkaz yüksek düzeyde tür olaylar ve bir örnek olarak enkaz sayacaktır unutmayın, beklenenden daha farklı bir yüzde GFP pozitif olayların olabilir. Bu bir sorun değildir. Ancak, daha fazla enkaz örnek, daha uzun sıralama alacak. </li><li> Deney ve analiz edilmesi gereken hücre tipine bolluğuna bağlı olarak, optimal verim veya optimal saflık sıralanmış hücreleri ya FACS hassasiyet modu ayarlayabilirsiniz. </li><li> RNA ekstraksiyonu için, uygun miktarda RNA ekstraksiyon tampon toplama tüpleri (1,5 ml mikrofuge&#39;de tüpler) hazırlar. Bu kurulum, 20.000 sıralama olaylar ekstraksiyon tamponu 350 ul (RNeasy mikro kiti, QIAGEN) sıralanır yaklaşık 100 ul toplam hacmi verecektir. Sıralama tamamlanmasının ardından hücre süspansiyonu üst kısmındaki havuz gibi örnekleri karıştırın. </li><li> Örnekleri saklayın veya RNA ekstraksiyon doğrudan devam. Başarılı mikroarray analizleri olduğunca az 500 sıralanır olaylar çıkarılan RNA preformed olmuştur. Burada, RNeasy mikro ekstraksiyon kiti (QIAGEN), WT-Ovation Pico RNA Amplifikasyon Sistemi ve FL-Ovation cDNA Biotin Modülü V2 (NuGEN) kullanılır. </li></ol> Temsilcisi Sonuçlar Yaklaşık 1.500 bir hafta eski P SCR Bir phytatray: 60.000 protoplast (hemasitometre tarafından ölçülen) hakkında GFP fide vermiştir. 65.000 FACS işlenmiş olaylar% 2.6 GFP-pozitif olarak tanımlanmıştır (Şekil 4b) sıralanır. Yaklaşık 1.500 dört gün eski P WOX5 sekiz tabak: GFP fideler her (toplam 12.000) 30.000.000 protoplast (hemasitometre tarafından ölçülen) hakkında vermiştir. 16.000.000 FACS işlenmiş olayların% 0.063 GFP-pozitif olarak tanımlandı ve sıralanır (Şekil 4c). 10.000 sıralanmış olaylar genelde RNA ekstraksiyonu için kullanılır ve 20 ile 140 ng toplam RNA (Şekil 5) verim alınabilir. <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="Şekil 1" /> Şekil 1. Arabidopsis kök hücre tipine özgü GFP işaretleyici hatları. Floresan mikroskobu görüntüleri diferansiyel girişim kontrast (DIC) ve Metamorph yazılımı (Moleküler Devices) üzerinde çalışan bir Eclipse 90i mikroskop (Nikon) GFP filtre ile çekildi. DIC ve GFP görüntülerin canlandırma amacı üst üste. Bu görsel deneyde kullanılan iki belirteç hatları gösterilir;a) P SCR: GFP ve b) P WOX5: GFP. <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="Şekil 2" /> Şekil 2. Bitki büyüme koşulları. Fideler phytatrays (a) veya dikey konumlandırılmış agar plakları (b) hydroponically bir çevre denetleyicisi yetiştirilmiştir. <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="Şekil 3" /> Şekil 3. Bitki protoplast GFP ifade Floresan mikroskobu görüntüleri diferansiyel girişim kontrast (DIC) ve GFP Metamorph yazılımı (Moleküler Devices) üzerinde çalışan bir Eclipse 90i mikroskop (Nikon) bir filtre ile çekildi. DIC ve GFP görüntülerin canlandırma amacı üst üste. Oklar bir patlama hücre, hücre artıkları ve GFP pozitif protoplast göstermektedir. Iki beyaz çizgiler arasındaki mesafe 50 mikron. <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="Şekil 4" /> Şekil 4. Floresan aktive GFP pozitif protoplast hücre sıralama vahşi tip (a) P SCR türetilen protoplast:: GFP (b) veya P WOX5: GFP (c) işaretleyici hatları bir FACSAria (BD) ile analiz ve sıralanmış tanımlanan kapıları kullanarak karşı kırmızı floresan (610/20 nm; y ekseni), yeşil (x ekseni 530 / 30 nm) bir dotplot. 100.000 olaylar her bir parsel sunulmaktadır. GFP ayırma kapısı giren olaylar yeşil vurgulanır. <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="Şekil 5" /> Şekil 5. 10.000 sıralanmış hücrelerden Temsilcisi RNA ekstraksiyon Hücreler RNA ekstraksiyon tamponu (QIAGEN) doğrudan sıralaması, RNA saflaştırılmış ve 2100 Bioanalyzer (Agilent), konsantrasyon, saflık ve bütünlüğü için kontrol edildi . Hem işaretleyici hatları gösterildiği için üç çoğaltır.

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

Protoplast, prensip olarak, elverişli koşulları optimize RNA kalitesi ve miktarı büyük ölçüde artıracak, çeşitli bitki dokularında elde edilebilir. Protoplasting çözüm ve seçmeli inkübasyon tampon kullanılan her ikisi de bu yönünü etkileyecektir. Birçok farklı floresan proteinleri kullanılan FACS yeteneklerine bağlı olarak, kullanılabilir, örneğin, GFP, RFP, YFP, CFP ya da birçok varyantları ve türevleri . Belirteçlerin ifade buradaki gibi sadece h?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
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fluorescence activated cell sorting of plant protoplasts

Yüksek çözünürlüklü, gen ekspresyonu hücre tipi özel analiz gelişim düzenleme ve herhangi bir çok hücreli bir canlının çevresel uyaranlara verilen yanıtlar anlayışı büyük ölçüde artırır. In situ hibridizasyon ve raportör gen görselleştirme kantitatif sınırlı bir ölçüde bu amaçla kullanılabilir ama yüksek çözünürlüklü RT-PCR veya özel hücre tiplerinden yüksek verim transcriptome çapında analiz RNA izolasyonu şarttır. Belirli bir hücre tipi ve sonraki Floresan Aktif Hücre Sıralama (FACS) floresan proteini işareti ifade doku Hücresel disosiyasyon mümkün RNA ekstraksiyon, cDNA sentezi / amplifikasyon ve mikroarray analizi için yeterli miktarda malzeme toplamak için yapar. Hücre tipi özel floresan muhabiri hatlarının geniş bir bitki araştırma topluluk için kullanılabilir. Bu durumda, Arabidopsis thaliana kök iki belirteç hatları kullanılır: P SCR: GFP (endodermis ve latent merkezi) ve P WOX5: GFP (latent merkezi). Fideler çok sayıda (binlerce) hydroponically veya agar plaklarına büyüdü ve daha fazla analiz için yeterli kök malzeme elde etmek için hasat edilir. Hücresel disosiyasyon bitki materyali hücre duvarının enzimatik sindirimi ile elde edilir. Bu prosedür yüksek ozmolarite indüklenen plasmolysis ve piyasada mevcut sellülazlar yapar, pectinases ve hemicellulases çözüm protoplast serbest bırakmak için. GFP pozitif hücreler FACS 488 nm lazer tarafından heyecan protoplast kırmızı emisyon spektrumları karşı yeşil görselleştirme yapar. GFP pozitif protoplast yeşil kırmızı salma artan oranı ile ayırt edilebilir. Protoplast genellikle doğrudan RNA ekstraksiyon tamponu içine sıralanır ve daha sonraki bir zamanda daha fazla işlem için saklanır. Bu teknik, basit ve uygulanabilir olduğu ortaya. Ayrıca, çok kıt hücre tipleri (örneğin latent merkezi hücreleri) için bile, transcriptome analizi için yeterli sayıda hücre izole etmek için zorluk olmadan kullanılabilir olması gösterilmiştir. Son olarak, Arabidopsis fide için bir büyüme kurulum (örneğin hücre tipi özel biyotik veya abiyotik stres tepkileri analiz için) hücre sıralama önce bitkilerin komplikasyonsuz bir tedavi sağlar gösterilmiştir . Potansiyel ek bitki protoplast FACS tartışılmıştır kullanır.

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

Bitki materyali belirli hücre türlerini izole etmek için kullanılan bir yöntem olduğunu göstermiştir. Bu teknik, özellikle hücre tipleri, hücresel bölünmeler ve Floresans Aktif Hücre Sıralama floresan proteinleri ifade transgenik işaretleyici hatları kullanmaktadır. Ayrıca, büyüme kurulumu kolaylaştıran tedavisi burada kuruldu<em&gt; Arabidopsis thaliana</em&gt; Hücre sıralama önce fide.

Bitki protoplast Floresan Aktif Hücre Ayırma

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

Research

JoVE Journal

Biology

24.7K Görüntüleme.  New York University. Bitki materyali belirli hücre türlerini izole etmek için kullanılan bir yöntem olduğunu göstermiştir. Bu teknik, özellikle hücre tipleri, hücresel bölünmeler ve Floresans Aktif Hücre Sıralama floresan proteinleri ifade transgenik işaretleyici hatları kullanmaktadır. Ayrıca, büyüme kurulumu kolaylaştıran tedavisi burada kuruldu Arabidopsis thaliana Hücre sıralama önce fide.

Video: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Biyoloji

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) Etiketleme ve Çözünmüş Organik Kültür bağımsız Kimlik sonraki Floresan Aktive Hücre Sıralama Karbon-aşağılayıcı Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Floresans Aktif Hücre Ayırma (FACS) Taze Dokular Normal ve Kanser ilişkili Fibroblastlar İzolasyonu

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Ozmotik Su Geçirgenliği Katsayısı (P Ölçme<sub&gt; F</sub&gt;) Küresel Hücreleri: Bir Örnek olarak İzole Bitki protoplastlan

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Bir Lüminesans Saat Reporter ile Arabidopsis Protoplasts transfekte içinde Sirkadiyen fenotipleri Hızlı Analizi

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Floresan Mikroskopi ile Stromule Frekansı görselleştirme

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Bir Gel Yüzeyinde Büyüyen Bitki kökleri Işık Sac Flüoresan Mikroskopi

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Manyetik aktif hücre stratejileri yalıtmak ve Sinovyal sıvı elde edilen mezenkimal kök hücre bir tavşan modelinden arındırmak için sıralama

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...