Bu projenin finansmanı Ulusal Bilim Vakfı hibe OCE1029607 (XM) ve MCB0702125 (MAM) ve Gordon ve Betty Moore Vakfı (MAM) tarafından sağlandı.

1. Su örneği işleme <ol><li> Filtre 1 mikron gözenek boyutlu membran filtreler aracılığıyla 10L çevre su büyük parçacıklar ve bacteriovores kaldırın. Bir damacana su süzüntü toplayın. </li><li> Transferi 3 4 ml taze hazırlanmış paraformaldehid çözüm (% 10 PFA) içeren steril Eppendorf tüpleri (50 ml) içine 36 ml süzüntü her. Hücreleri korumak için 2 saat oda sıcaklığında inkübe edin. Vakum filtrasyon ile 0.22 mikron-gözenek boyutlu beyaz membran filtreler üzerine hücreleri toplayın. Vakum filtrasyonu filtre üzerinden geçen 10 ml salin (PBS) ve fosfat tamponlu filtre yıkayın. Negatif kontrol olarak filtreleri Etiket ve -20 ° C&#39;de saklayın </li></ol> Adımlar 1,3-1,4 DOC sınırlı koşullar oluşturulması için isteğe bağlıdır. <ol start="3"><li> (5 mcM NH 4 Cl, 5 mcM NaNO 3, ve 1 mikrona NaH 2 PO 4 final konsantrasyon) damacana inorganik azot ve fosfor karışımı ekleyin. </li><li> Zaman zaman ajitasyon ile 48 saat boyunca yerinde sıcaklığında karanlıkta inkübe edin. </li></ol> 2. Microcosm kurulması ve inkübasyon <ol><li> Her altı 1-L cam şişe, microcosms kurmak için adım 1.4 damacana 800 ml su numunesi ile doldurun. Her mikrokozmosu BrdU (10 mcM, nihai konsantrasyonu) ekleyin. İyice karıştırın. </li><li> Microcosms üç gruba 1 ml modeli DOC bileşik çözüm, bu DOC değişiklikler olarak görev yapacak. Kalan üç microcosms içine 1 ml steril PBS, bu ek kontrol olarak hizmet verecek. </li><li> Bir kuluçka çalkalayıcı microcosms inkübe ve 100 rpm&#39;de sallayarak sırasında in situ sıcaklığında karanlıkta inkübe edin . </li><li> Her mikrokozmosu ve transfer süresi 0 puan, 8, 16, ve 24 saat için 50 ml steril Eppendorf tüpleri içine 36 ml su numunesi toplayın. Hemen taze PFA toplama tüpleri 4 ml (% 10) ve hücreleri korumak için 2 saat oda sıcaklığında inkübe edin. </li><li> 0.22-mikron gözenek boyut polikarbonat membran filtreler aracılığıyla Filtre hücreleri korunmuş. Filtreleri 10 ml PBS ile yıkayın. Hemen bir sonraki adıma geçin veya -20 ° C&#39;de filtreleri saklamak </li></ol> 3 BrdU Ortaklığı yerinde İmmuno <ol><li> Oda sıcaklığında filtreler (DOC değiştirilmiştir numuneleri, herhangi bir ek kontrol ve negatif kontrol) Çözülme. </li><li> 1 ml lizozim çözümü uygulayın [10 mg / ml lizozim yumurta beyazı 100 mM Tris, 50 nM EDTA (pH = 8)] filtre bakteri hücreleri kapsayacak şekilde 4. 30 dakika oda sıcaklığında inkübe edin. Emme altında 10 ml PBS üzerinden geçerek filtre yıkayın. </li><li> 1 ml proteinaz K çözüm [2 mg / ml proteinaz K 100 mM Tris, 50 nM EDTA (pH = 8)] filtre bakteri hücreleri kapsayacak şekilde 4. 30 dakika oda sıcaklığında inkübe edin. Emme altında 10 ml PBS üzerinden geçerek filtre yıkayın. </li><li> 1 ml eksonükleaz çözüm [eksonükleaz III (50 U / ml) 5 mM MgCl 2 ve 50 mM Tris-HCl] 5 filtre bakteri hücreleri kapsayacak şekilde ekleyin. 37 ° C&#39;de 30 dakika. Emme altında 10 ml PBS üzerinden geçerek filtre yıkayın. </li><li> Üreticinin talimatlarına göre çerçeve mühür inkübasyon odaları (Bio-Rad) birleştirin. </li><li> Alkol-kuru temizleme yüzey steril bir bıçak kullanarak sekizde içine filtre dilimleyin. </li><li> Forseps kullanarak, bir monte çerçeve mühür inkübasyon odası (sekiz çerçeve mühür odaları, her filtre örnek için gerekli olan) bir filtre sekizinci bölümüne yerleştirin. Hücreleri içeren filtre bölümü tarafında yukarıya doğru bakmalıdır. Filtrenin arka kısmında nem filtresi sopa slayt sağlayacaktır. Filtre kuru olursa, slayttaki filtre bölümüne yerleştirmeden önce, odanın merkezinde dih 2 O küçük bir damla (2 ul) geçerlidir . </li></ol> Adımlar 3,8-3,20 üreticinin talimatlarına modifiye prosedürler izlenerek, Situ Hücre Çoğalması Kit ROCHE reaktifler FLUOS kullanabilirsiniz. PBS için hariç, tüm reaktifler kit içinde sağlanır. <ol start="8"><li> Mühür uygulandıktan sonra taşma neden olmaksızın eşit kuluçka odasının tüm filtre bölümünde karşılamak için yeterli inkübasyon tampon (% 0.5 sığır serum albümin, PBS içinde% 0.1 Tween20,) uygulayın. </li><li> Kuluçka odasının çerçeve üzerinde polyester çerçeve kapak yerleştirin. Kuluçka odasının mühür için sıkıca bastırın. Filtre üzerindeki hava kabarcıkları kaçının. Kapalı odaları karanlıkta, oda sıcaklığında 10 dakika inkübe edin. </li><li> Polyester mühür çıkarın ve inkübasyon odaları açmak. (Not: odaları açılması bazen slayt çerçeve mühür yukarı çekin Bu durumda, aşağıdaki adımları için yeni bir oda hazırlamak.) </li><li> Odanın bir köşesinde, kuluçka tampon dışarı pipetleyin. Filtresi ile temasından kaçının. </li><li> Hafifçe 100 ul PBS pipetleme filtreyi yıkayın ve3 kez odasından dışarı. </li><li> Kullanımdan hemen önce, üretici tarafından önerilen adımları izleyerek anti-BrdU-FLUOS çalışma çözeltisi hazırlayın. </li><li> Pipetleyin 120 ul anti-BrdU-FLUOS, filtre üzerine çözüm eşit olarak tüm yüzeyini kaplayan özen gösterin. </li><li> Odasına yeni bir polyester kapağını kapatın. Filtrenin üstünde hava kabarcıkları kaçının. 37 karanlık oda ° C&#39;de 3 saat. Bu adım, floresein izotiyosiyanat (FITC) ile in situ BrdU dahil DNA etiket. </li><li> Polyester kapağını çıkarın ve kuluçka odasının açık. Anti-BrdU-FLUOS çözüm pipetleyin. PBS ile filtre 3 kere yıkayın. </li><li> Kuluçka odasının steril bir yüzey için filtre aktarın. Steril bir bıçak kullanarak filtre bölümüne küçük parçalar halinde dilimleyin. </li><li> 2 ml mikrosantrifüj tüpler, 1 ml PBS içeren her filtre parçaları aktarın. Sıkıca parafilm ve mühür ile tüp kap. 37 ° ° C&#39;de ve 10 dakika için 200 rpm. </li><li> 5 dakika maksimum hızda vortexer ve girdap üzerine tüpleri sabitleyin. 15 ml steril Eppendorf tüp içine pipetleyin süpernatantı. 5 kez daha inkübasyon ve vorteks adımları tekrarlayın. Her numune için aynı 15 ml Eppendorf tüp süpernatant birleştirin. Tipik olarak,% 80 hücre süspansiyonu telafi edilebilir. </li><li> Süpernatant Mağaza yeniden süspanse hücreleri ile 4 ° C 2 gün içinde sıralayın. </li></ol> 4. FACS analizi BD FACSAria akış sitometresinin ve ilgili yazılım için bir prosedür burada tanımlanmıştır. <ol><li> Üretici tarafından önerilen akış sitometresi aşağıdaki adımları ayarı optimize edin. Bu ısrar ediyor: gerekli kopmasına parametreleri ve tatlı nokta ayarlamak için amplifikasyon kontrolü ayarlayarak; deneme kılıf basınç için lazer gecikme ve alan ölçekleme faktörleri optimize edilmesi ve FSC ve SSC gerilimler, FSC eşik, FSC floresan ölçekleme, floresan kompansasyonunu ayarlarını optimize , vb. </li><li> FITC ve yan dağılım (SSC) floresan yoğunluğuna göre akış sitometresi (FCM) negatif kontrol örnekleri çalıştırın. FITC eşik negatif kontroller hiçbir hücre FITC SSC satın ekran görüntülenebilmekte kadar artırın. </li><li> Herhangi bir ek kontrol numunelerinin çalışılabilmesi ve FITC ve SSC satın alma dayalı 10.000 hücrelerinin dağılımı desen inceleyin. Tüm hücrelerin içine ve &quot;düşük yoğunluklu hücreleri&quot; (Lis) olarak belirlemek için bir kapı tanımlayın. </li><li> DOC-tadil örnekleri çalıştırın ve FITC ve SSC dayalı 10.000 hücrelerinin dağılım paterni incelemek. Bazı hücreler, önceden belirlenmiş LI kapısında belirir. Lis daha yüksek floresan (HIS) hücrelerin içine başka bir kapı tanımlayın. Kapılı hücrelerin göreceli bolluk görüntülemek istatistiklerini edinin. </li><li> &quot;Arındırır 1 damla&quot; modunda 500 ul PBS içeren toplama tüpleri içine sırala HI hücreleri. Onun sayısı 500.000 sayar ulaştığında sıralama sonlandırın. </li></ol> 5. Filtre PCR 16S rRNA genleri Filtre PCR işlemleri Kirchman ark değiştirilmiş 6 <ol><li> Filtre beyaz üzerine sıralanmış hücreleri, 0.22 mikron gözenek boyutu, 25 mm çap, polikarbonat membran filtreden. Steril bir bıçak kullanarak herhangi bir bakteri hücreleri içeren filtre kenarından kesin. Filtre 8 eşit büyüklükte parçalar halinde dilimleyin. </li><li> Hücreleri tüp içe bakan, bir PCR reaksiyonu tüp içine tek bir filtre parçası yerleştirin. PCR reaksiyonu tüpe 45 ul PCR sınıf su ekleyin. Tamamen su filtre Submerge. </li><li> 2 ROCHE versiyonu gösterime PuReTaq okuyun-To-Go, PCR reaksiyonu tüpe PCR Boncuk kısaca vorteks ekleyin. PCR reaksiyon tüpleri her birine 2 ul 27F ve 1492R 7 olarak ileri ve geri 16S rRNA gen primerleri (0.4 mcM her astar için son konsantrasyon), her ekleyin. </li><li> (Isteğe bağlı) adsorbe amplifikasyon inhibitörlerinin yardımcı olmak için PCR reaksiyon karışımının 1μl sığır serum albumin solüsyonu (BSA, nihai konsantrasyonu 30μg/100 ul) ekleyin. BSA eklemek seçerse, bunun yerine su 1μl eklemek. </li><li> Termal cycler PCR gerçekleştirin. PCR programı aşağı bir dokunuş tavsiye edilir, hangi sırayla 62 ve 52 azalan tavlama sıcaklığına sahiptir ° C 1 ° C 11 52 tavlama sıcaklığı 15 döngüleri takip döngüleri, ° C için çevrim başına Tüm döngüleri, denatüre (95 ° C), (62 52 ° C) tavlama ve uzatma (72 ° C&#39;de) 50&#39;li süresi adımlarla içerir. Bir ilk 3 dakika denatürasyon ve son 10 dakikalık uzatma adım PCR programı da dahil. </li><li> Etidyum bromür lekeli% 1 agaroz jel elektroforezi ile PCR onaylayın. Vergi PCR jel amplikonlarının ve QIAGEN QIAquick jel ekstraksiyon kiti temiz. </li><li> Her numune için iki ek PCR gerçekleştirin, her zaman yeni bir filtre bölümünü kullanabilirsiniz. PCR jel purificati sonraüzerinde, birlikte aynı örnek amplikonlarının havuzu. Saf 16S rDNA amplikonlarının klon kütüphane yapım ve sıralama olarak taksonomik tanımlama, izin veren bir dizi moleküler analizler için hazır. </li></ol> 6. Temsilcisi sonuçları: Temsilcisi sonuçlar bir örnek olarak putrescine aşağılayıcı bakteri bir çalışma ile açıklanmıştır. Gürcistan sahil kesiminde yer alan su örnekleri alınan ve yukarıda açıklanan prosedürleri takip işlendi. FACS analizi putrescine Ayrıca yüksek BrdU birleşme oranı (Şekil 1) gösteren, yüksek FITC floresan bir grup bakteri gelişimi neden olduğu saptandı. Bu hücreler yüksek BrdU dahil hücreleri (HIS) olarak belirlenmiş ve çoğunlukla putrescine-aşağılayıcı bakteri içermesi bekleniyor. Onun sadece düşük düzeyde BrdU dahil (LIS) ile hücreler içeren herhangi ek kontroller, eksikti. Lis esas eklendi putrescine kullanmak için koyamadık bacterioplankton içeren bekleniyor. Onun ayrı tüpler içine sıralanır ve sonra membran filtreleri üzerine toplandı. 16S rRNA gen amplikonlarının filtre PCR kullanarak yüksek HI hücreler için elde edilmiştir. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Şekil 1"/> Şekil 1. ek kontrolü ve inkübasyon 24 saat sonra toplanan, (Burada bir örnek olarak putrescine) model bileşik-tadil örnek Akış sitometrik analizi . Hücre dağılımı analizi (1) olumlu BrdU dahil düzeyi ile ilgili FITC etiketleme (x-ekseni), floresan dayalı, ve (2) yan dağılım (SSC, y-ekseni), olumlu bir hücre ile ilgili olduğu boyutu. Kapısı gösterimde BrdU dahil, (LI, düşük BrdU-birleşme HI yüksek BrdU dahil) düzeyi dayanmaktadır. HI ve LI hücrelerin göreli yüzdesi ilgili kapıları gösterilmiştir.

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Çıkar çatışması ilan etti.

Bizim yaklaşımımız çiftler BrdU bir FACS ve 16S rDNA analiz sucul ortamlarda bireysel DOC bileşenleri metabolize bacterioplankton tür düzeyinde kimlik izin vermek. BrdU dahil tahlil bakteri hücrelerinin metabolik faaliyetleri, sadece aktif bakteri analizi sağlar ve böylece uyuyan hücreleri içermez dayalı etiketler. Bizim yaklaşımımız, BrdU dahil, sonradan görselleştirildiği BrdU esas farklı düzeylerde immunodetected yerinde ve bakteri FACS kullanarak gruplandırılmış ve sıralanmış. ...

<table border="1"><tbody><tr><td> Isim </td><td> Tip </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> BrdU </td><td> Reaktif </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lizozim </td><td> Reaktif </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinaz K </td><td> Reaktif </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> Situ Hücre Çoğalması Takımı, FLUOS </td><td> Seti </td><td> Roche </td><td> 11810740001 </td><td> Anti-BrdU-FLUOS ve üretici tarafından önerilen daha reaksiyon başına Kuluçka tampon daha fazla tüketin. </td></tr><tr><td> Çerçeve-Seal Kuluçka Odaları </td><td> Malzeme </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> Polikarbonat Membran Filtreler (142 mm çap, 1,0 mikron gözenek boyutu) </td><td> Malzeme </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Polikarbonat Membran Filtreler (25 mm çap, 0,2 mikron gözenek boyutu) </td><td> Malzeme </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> versiyonu gösterime PuReTaq Ready-To-Go PCR boncuklar </td><td> Seti </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick jel ekstraksiyon kiti </td><td> Seti </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR Sistemi </td><td> Seti </td><td> Epicentre </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Mikroplar sucul ortamlarda pek çok çözünmüş organik karbon (DOC) yüzeylerde bozulması aracılık önemli ajanlardır. Ancak, doğada DOC özel havuzları dönüştürmek bakteri taksonların belirlenmesi teknik bir sorun teşkil etmektedir. Burada bir yaklaşım tarif çiftler bromodeoxyuridine (BrdU) dahil, floresan aktif hücre sıralama (FACS) ve 16S rRNA gen tabanlı sucul ortamlarda aşağılayıcı belirli bir DOC bileşik yetenekli bacterioplankton kültür-bağımsız bir kimlik sağlar moleküler analizler. Üç kat bacterioplankton microcosms BrdU ve bir model DOC bileşik (DOC değişiklikler), ya da sadece BrdU (no ek kontrol) almak için ayarlanır. Yeni sentezlenmiş bakteriyel DNA ve BrdU-işaretli DNA timidin BrdU yerine pozisyonları kolayca immunodetected 1,2 olabilir. Katma DOC bileşik seçici aktive olması beklenen ve bu nedenle hiçbir BrdU esas DOC değişiklikler ve hücre dışı duyarlı hücreler daha yüksek seviyelerde (HI hücreleri) vardır kullanabilmek 24 saat inkübasyon, bacterioplankton sayesinde Ayrıca kontroller (düşük BrdU dahil hücreleri LI hücreleri). Floresan İmmuno sonra, HI hücreleri ayırt edilirler ve fiziksel floresan aktive hücre sıralama (FACS) 3 LI hücreleri ayrılır. Sıralama DOC-duyarlı hücreleri (HI hücreleri) DNA ayıklanır ve taksonomik rRNA gen-temelli analiz PCR, klon kütüphane yapım ve sıralama da dahil olmak üzere sonraki 16S ile tanımlanır.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

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

Çevre bacterioplankton bir model ile çözünmüş organik karbon (DOC) bileşik ve bir DNA etiketleme reaktif bromodeoxyuridine (BrdU) inkübe edilir. Daha sonra floresan aktif hücre sıralama (FACS) kullanarak kendi yükseltilmiş BrdU dahil dayalı toplu topluluk, DOC-aşağılayıcı hücreleri ayrılır. Bu hücreler daha sonra sonraki moleküler analizleri ile tespit edilir.

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 ...

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Research

JoVE Journal

Biology

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

20.8K Görüntüleme.  Kent State University. Çevre bacterioplankton bir model ile çözünmüş organik karbon (DOC) bileşik ve bir DNA etiketleme reaktif bromodeoxyuridine (BrdU) inkübe edilir. Daha sonra floresan aktif hücre sıralama (FACS) kullanarak kendi yükseltilmiş BrdU dahil dayalı toplu topluluk, DOC-aşağılayıcı hücreleri ayrılır. Bu hücreler daha sonra sonraki moleküler analizleri ile tespit edilir.

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biyoloji

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

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 ...

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 ...

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 ...

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 ...

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 ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Ultrastrüktürel Analizler için Maya endozomal Sisteminin Nanogold Etiketleme

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases ve Kofaktör Analoglarının ile Nükleik Asitler ve Proteinler Dizi-spesifik Etiketleme

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Dev Virüs Karışım Ayırma için Floresan-Aktive Hücre Sıralama alternatif bir strateji olarak Tek Hücreli Mikro-aspirasyon

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...