Biz FACS sıralama ile yardım için Dr Yitzchak Oschry ve Dr Orit Sagi-Asif ederim. Bu araştırma hibe sözleşmesi n kapsamında Avrupa Birliği Yedinci Çerçeve Programı (FP7/2007-2013) NE hibe tarafından desteklenen ° [276.890], İsrail Kanser Derneği (# 20110078), gelen ve İsrail Kanser Araştırma Fonu (Research Kariyer Geliştirme Ödülü).

1. Fare gelen Meme veya Deri Doku Diseksiyon <ol><li> Farelerin istenilen doku diseksiyon önce, aşağıdaki sarf malzemeleri ve reaktifler hazırlamak: Malzemeleri: <ul><li> Cerrahi araçları (deterjan ile yıkandı ve daha sonra% 70 etanol içinde batırılmış). </li><li> Strafor yüzey diseksiyonu sırasında fare dinlenmek için. </li><li> Veya iğne diseksiyonu sırasında fare tutun. </li><li> Manyetik karıştırıcı (otoklav) içeren sindirim için kapaklı cam kavanoz,. </li><li> 37 Su banyosu ° C, heyecan plaka üzerinde dinlenirken, bir dalgıç manyetik karıştırıcı ve batığın sindirim kavanozu için yeterli miktarda su içeren. </li></ul> Reaktifler: <ul><li> FACS tampon I: (4 ° C): Dulbecco Fosfat (Kalsiyum Magnezyum Ücretsiz) Salin CMF Buffered +% 0.5 BSA </li><li> Kollajenaz çözeltisi (kullanımdan hemen önce yapılmış): 0.05 g kollajenaz II 0.05 g kollajenaz IV 0.01 g deoksiribonükleaz 20ml FACS tampon I. kullanıma kadar buz üzerinde kollajenaz çözüm tutun. </li><li> PharmLyse (Amonyum Klorür Parçalama Reaktifi). </li><li> FACS tampon II: Dulbecco&#39;nun Fosfat Tamponlu Tuzlu CMF +% 1 FCS. </li></ul></li><li> Meme bezlerinin diseksiyonu (ayrıca 17 bakınız). <ul><li> Karbon dioksit (CO 2) inhalasyon kullanmaya dişi farelerde Euthanize. </li><li> Strafor yüzey üzerinde, sırtında fare yerleştirin ve sıkıca 25 gauge iğneler (Şekil 1A) kullanarak tüm dört bacağı pin. % 70 etanol ile cömertçe fare püskürtün. </li><li> Forseps kullanarak, orta hatta karın cildi yukarı çekin ve keskin bir makas ile küçük bir kesi yapmak. </li><li> Abdominal veya torasik kavite delinme kaçınarak kesi başlayarak, hayvanın boynuna deri yukarı kesti. </li><li> &quot;Y şekli&quot; ile sonuçlanan, bacak karşı orta hattan bir kesik, arka bacaklar üzerine deri kesin. </li><li> Fare vücuttan uzakta deri çekin ve aşağı pin, teşhirsalgı derinin alt (Şekil 1B) tutturulmuştur. </li><li> Fare omurganın doğru meme bezi ayırmak için küçük keskin bir makas ile katılgandoku kesim sırasında cildi nazikçe gelen meme bezleri ayırmak için Q-ipuçlarını kullanın. </li><li> Karın bezleri (# 4, 5) Bu protokol için en iyisidir. Ayrıca 2. meme bezi kullanabilirsiniz, ancak bu bezlerin anatomik karışık dokusu kaynaklı hücreler neden ayırmak zor olabilir pektoral kas yakın bulunan aklınızda olabilir. </li><li> Not: Tümör dokusunda diseksiyon, bu nekrotik bölgelerin çıkarılması tavsiye edilir. </li></ul></li><li> Cilt dokusunun Diseksiyonu: epilasyon ile uğraşmak zorunda kalmadan, cilt dokusu elde etmek için en kolay yolu fare kulakları kullanmaktır. Doku büyük miktarda gerekiyorsa, fare gövde saç kapalı tıraş ve cilt teşrih edebilirsiniz. <ul><li> CO 2 kullanarak farelerde Euthanize inhalation. </li><li> Keskin bir makas kullanarak, buz PBS küçük hacimli (~ 0.5 ml) içeren sindirim kavanoza hemen kulakları ve yer hem kesti. </li></ul></li></ol> 2. Meme Bezi / cilt Tek Hücre Süspansiyon hazırlanması <ul><li> Meme bezlerinin / buz PBS küçük bir hacim içeren sindirim kavanoza cilt yerleştirin. Doku sonra PBS ile kanlı yıkama x2 ise ve aşırı PBS atın. </li><li> Kavisli makas ile iyice kıyın. </li><li> 20 ml kollajenaz çözüm (Not: kollajenaz çözüm bu miktar verimli 15 fareleri kadar gelen meme veya tümör dokusunun ve kulakları yaklaşık 5 g ayrıştırmaları) ekleyin. </li><li> 37 plaka karıştırın üzerine hemen yerleştirin ° 15 dakika C su banyosu, ve inkübe orta hızda karıştırmaya devam. </li></ul> Not: diğer dokuları sindirerek, optimum inkübasyon sürelerinde farklılık gösterebilir. <ul><li> Reaksiyon durdurmak için, 30 ml soğuk DMEM +% 10 FCS ekleyin. </li><li> Stra50 ml&#39;lik bir konik boru üzerine yerleştirilmiş bir 70 mikron hücre süzgecinden doku / medya karışım halinde. </li><li> 4 az 450 x g&#39;de 5 dakika boyunca konik bir tüp Santrifüj ° C. </li></ul> 3. Kırmızı Kan Hücreleri Lizis Not: öldürülmeden önce fareler PBS ile kalp-perfüze olsaydı bu adım gerekli değildir. <ul><li> Hücre peleti bozmadan, vakum bağlı bir cam pipet kullanılarak süpernatan aspire. </li><li> Oda sıcaklığında 5 dakika boyunca 10 ml PharmLyse çözelti ve inkübe içinde RBC, tekrar süspansiyon hücre peleti lyse. </li><li> FACS tampon I&#39;in yaklaşık olarak 40 ml ilave edilerek PharmLyse çözelti Nötrleştir </li><li> 4 ° C&#39;de 450 x g&#39;de 5 dakika için hücreler ile konik bir tüp döndürme </li><li> Süpernatant aspire. </li></ul> 4. Endojen Fc Engelleme <ul><li> İsteğe bağlı: Aşağıdaki adımlar için uygun hacmini belirlemek için sayım hücreleri. </li><li> Hücreler süspanse1-3 ml FACS tampon I (hücre sayısına bağlı olarak, ve kullanmak istediğiniz antikor miktarı). </li><li> 1:50 seyreltme (10 ug / ml) ulaşır, ve 10-20 &#39;için buz üzerinde inkübe FcBlock (anti-fare CD16/CD32) ekleyin. Hayır FcBlock yıkayın gerekir. </li></ul> 5. Lekelenme <ul><li> Un-lekeli kontrolü ve tek renk denetimleri (kullanılan floresan antikor sayısına göre) olarak kullanılmak üzere Eppendorf tüplere 100 ul hacimde çıkarın. </li><li> Etiket fibroblastlar için: 1:50 seyreltme (10 ug / ml) ile (florofor ile direkt olarak konjuge) anti-PDGFRα ekleyin. </li><li> Diğer hücre popülasyonlarının (örneğin bağışıklık hücreleri, makrofajlar, endotel hücreleri vb) etiketlemek için: 1:50 seyreltme, yüzey belirteçleri için uygun floresan konjuge antikor ekleyin. </li></ul> Not: PDGFRα fibroblastlar için bir işaretleyici güçlü olmasına rağmen, bağışıklık hücreleri için ve ord epitel hücreleri için antikorları içerir tavsiye edilirer herhangi bir çift pozitif nüfus dışlamak ve dolayısıyla izole fibroblastların saflığı geliştirmek için. <ul><li> Tek bir renk kontrol eppendorf tüpleri her birinin her bir antikorun 2 ul ekle. </li><li> Karanlıkta buz üzerinde 1 saat için antikorlar ile hücreler inkübe edin. </li><li> Yıkamak için: FACS tampon I tüpleri doldurmak ve 4 ° C&#39;de 450 x g&#39;de 5 dakika daha dönmeye </li><li> Yaklaşık olarak 2 x 10 6 hücre / mL, veya hangisi uygun FACS sıralayıcı ile sıralama için en uygun konsantrasyon arasında bir konsantrasyonu için bir FACS buffer içinde süpernatant, tekrar süspansiyon hücreleri aspire. </li></ul> İsteğe: Hücreler bir sıralama süre için FACS tampon II içinde yeniden süspanse edilebilir. Bu hücre canlılığı artırabilir. Bununla birlikte, serum içinde faktörlere maruz kalma, test edilmiş ve göz önüne alınması gereken bir gen ekspresyonunu etkileyen sahip olabilir. <ul><li> Hücre kümeleri önlemek için tüpleri içine filtre hücreleri: Filtre üst FACS tüpler içine etiketli hücreleri aktarın. </li><li> Ölü hücre dışlamak içins, eklenti 7AAD (0.5 mg / ml) veya örnekleri (un-lekeli kontrolü hariç) Propidium İyodür (PI). </li></ul> Not: hücre kaybını önlemek için, FACS kaydettikten sonra un-lekeli örnek 7AAD/PI eklemek ve 7AAD-tek kontrol yeri olarak tekrar kullanabilirsiniz. <ul><li> Analiz ederken buz örnekleri tutun. </li></ul> 6. FACS tarafından Hücre İzolasyonu (Not: protokol bu bölümü bir FACS sıralayıcı, örn. BD FACS AriaII, ya da bir teknisyen yardımı işletim önceki bilgi gerektirir). <ul><li> FACS sıralayıcı yazılımı (BD FACSDiva) kullanarak ileri saçılım (FSC), yan dağılım (SSC), 7AAD, FITC, PE ve etiket hücreleri (Şekil 2) için kullanılan herhangi bir fluorofor analiz etmek için uygun analiz araziler hazırlar. </li><li> Hücre sıralayıcı her örnek yüklemeden önce, vorteks kısaca hücreleri tekrar süspansiyon. </li><li> Veya boyanmamış numune analiz ederek başlayınder hücre artıkları dışarı kapısına, total hücre nüfus için yolluk ayarlamak ve otofloresans veya arka plan boyama belirlemektir. </li><li> Toplam hücre popülasyonunun canlı hücre nüfusu belirlemek ve kapısı unstained örnek + 7AAD analiz edin. </li><li> Tazminat gibi parametrelerin belirlenmesi ve kalibre etmek için her bir renk kontrolü analiz edin. </li><li> Sıralama için kapılarının konumunu tanımlamak için lekeli numune analiz edin. </li><li> Sonra istenilen hücre popülasyonlarının için parametreler ve kapıları set edilmiştir, Eppendorf tüpler veya kültür ortamı veya RNA lizis tamponu (örneğin RLT tampon veya Trizol gibi) içeren 15 ml konik tüpler içine etiketli hücre popülasyonu sıralama başlar. </li></ul> Not: hücreler, çok sayıda ayırma, bu hücreler lizis tamponu ile seyreltik verimi düşer eşlik eden kılıf sıvının büyük bir hacim olarak, RNA lizis tamponu içine direkt olarak sıralamak için tavsiye edilmez. <ul><li> Immediat aşağı Spin hücreleriely sonra sıralama bitmiş ve deneme amacına bağlı olarak taze orta veya RNA lizis tamponu, içine transfer edilir. </li><li> Kültürü izole hücreler için sıralama ise, steril sıralama yapmak. Hücrelerin daha iyi bir iyileşme için, fenol kırmızısı ücretsiz DMEM +% 5 FCS yerine FACS tampon II kullanımı ve% 20 FCS ile kültür ortamında hücreleri toplamak. </li><li> Kültür fibroblastlar kriteri ise, kendi gen ekspresyonu değişiklikleri neden fibroblast aktivasyonunu bu sonuç olarak plastik Kollajen kaplı plakalar üzerinde alwaysplate ve asla doğrudan. </li></ul>

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Çıkar çatışması ilan etti.

Doku kültüründe gerçekleştirilen deneyler bilgilendirici ve in vivo olarak kontrol edilebilir fonksiyonel ilkeler önermek mümkün olmakla birlikte, bu kültür içinde 11,12 hücrelerde gen ekspresyonunu büyük değişiklikler meydana geldiği bilinmektedir. Fibroblastlar gen ekspresyonu sırasında bir doku kültürü adım önlemek için amacıyla, sağlar taze fare veya insan dokusu normal hem de kanser-kaynaklı fibroblastlann izole edilmesi. Bir protokol gelişmiş Bu protokol başarıyl...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strongReaktif&gt; İsim</strong</td&gt;<td<strong&gt; Şirket</strong</td&gt;<td<strong&gt; Katalog numarası</strong</td&gt;<td<strong&gt; Yorumlar (isteğe bağlı)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco</td&gt;<td&gt; 41.965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Biyolojik Sanayi</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Kollajenaz II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Kollajenaz IV</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Deoksiribonükleaz</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555.899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Hücre süzgeç 70 mikron</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Saflaştırılmış anti-fare CD16/CD32</td&gt;<td&gt; BD Pharmingen</td&gt;<td&gt; 553.142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Via prob (7AAD)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-fare CD140a-PE (PDGFRA)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-fare F4/80- FITC</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM w / o Fenol Kırmızısı</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Kollajen Tip I</td&gt;<td&gt; BD Biosciences</td&gt;<td&gt; 354.236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Kanser ile bağlantılı fibroblast (cafs) özellikle göğüs kanseri olmak üzere birçok kanser tümör stroma içinde en önemli hücre türüdürler ve bunların belirgin durum çoğu zaman zayıf prognoz 1,2 ile ilişkilidir. Cafs stromal fibroblastlann bir aktive alt gruptaki olup, bunların çoğu bu myo işaretleyici α-SMA 3 ifade eder. Cafs lokal doku fibroblast yanı sıra kemik iliği kökenli hücrelerin gelişen tümör içine alınmış ve tümör mikro 4 etkisi altında CAF fenotip kabul kaynaklanır. Cafs tümör hücre proliferasyonu, anjiyogenez ve işgali 5-8 teşvik eden sinyal yoluyla tümör gelişimini, büyümesi ve ilerlemesi kolaylaştırmak için gösterildi. Bu cafs erken ön-neoplastik aşamaları 9 başlayarak, tümör terfili iltihap aracılık ederek tümör büyümesini artırmak olduğunu göstermiştir. Anahtar rol cafs rağmen artan kanıtlar, tümör büyümesini kolaylaştırmada oynamakokuyan cafs tümör mikro 10 co-mevcut pek çok alt tipi ile CAF-spesifik belirteçler ve bu hücrelerin büyük heterojenliğin eksikliği nedeniyle devam eden bir meydan olmuştur. Üstelik, in vitro olarak fibroblastlar okuyan bunların gen ekspresyonu profiline sıkça doku kültürü 11,12 değiştirildiği gerçeği ile engellenmektedir. Bu sorunu çözmek için ve taze fare ve insan dokularından fibroblastların tarafsız gen ekspresyon profili izin, biz Floresans-Aktif Hücre Ayırma (FACS) 13,14 için daha önceki protokollere dayalı bir yöntem geliştirdi. Bizim yaklaşımımız taze fare ve insan dokusundan fibroblastlar yalıtmak için bir yüzey işaretleyici olarak PDGFRα kullanarak dayanır. PDGFRα bol, normal fibroblastlar ve cafs 9,15 ikisi tarafından ifade edilir. Bu yöntem de dahil olmak üzere, normal fibroblastlar ve cafs saf popülasyonlarının, izolasyonu ancak α-SMA sınırlı sağlar + miyofibroblastlar aktif. İzole fibroblastlar sonra olabilirkarakterizasyonu ve tümörogenez boyunca cafs meydana gen ifadesinin evrim karşılaştırma için kullanılır. Nitekim, biz ve diğerleri 16 sıralama hücre izole fibroblastların ekspresyon profili bildirdi. Bu protokol başarıyla deri, meme, pankreas ve akciğer dokularından fibroblastların zenginleştirilmiş popülasyonları ayırmak ve profil yapıldı. Bunun yanı sıra, yöntem, aynı zamanda işlevsel deneyler için ve kültür cafs çalışırken sık sık büyük bir engel teşkil tümör hücreleri ile kirlenmesini önlemek üzere, sıralı hücrelerin kültürlenmesi izin verir.

Doku fibroblast zenginleştirilmiş popülasyonlarının izolasyonu fibroblastlar sonuçları için bir belirteç olarak PDGFRα kullanma. Sıralama sonrasında saflık düzeyi gibi post-tür analizi (Şekil 2A) ile ölçülebilir,% 99 idi. Hücre özel kontrol genlerinin göreceli ifadesi (Şekil 2B) ile non-fibroblast hücreleri kirletici yüzdesi tahmin edilmesi tipik olarak% 0.1-0.6 kirlenme göstermektedir. Saflık Bu düzeyde izole fibroblastlar 9 yüksek kalitede trans...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

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

Kanser İlişkili Fibroblastlar (cafs) proliferasyon, anjiyogenez ve inflamasyon teşvik eden sinyal yoluyla tümör gelişimini, büyüme ve ilerlemesini hızlandırabilir. Burada bir yüzey işaretleyici olarak PDGFRα kullanarak hücre sıralama ile taze fare ve insan dokularında normal fibroblastlar ve cafs saf popülasyonlarının izole etmek için bir yöntem açıklanmaktadır.

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 ...

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Research

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Biology

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

35.5K Görüntüleme.  Tel Aviv University. Kanser İlişkili Fibroblastlar (cafs) proliferasyon, anjiyogenez ve inflamasyon teşvik eden sinyal yoluyla tümör gelişimini, büyüme ve ilerlemesini hızlandırabilir. Burada bir yüzey işaretleyici olarak PDGFRα kullanarak hücre sıralama ile taze fare ve insan dokularında normal fibroblastlar ve cafs saf popülasyonlarının izole etmek için bir yöntem açıklanmaktadır.

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

İzolasyon ve Arıtma Drosophila</emManyetik Boncuk Sıralama> Periferik Nöronlar

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

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 ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Sıçan Portal Fibroblastlar izolasyonu ile<em&gt; Yerinde</em&gt; Karaciğer Perfüzyon

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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 ...

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

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

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

Spektral Sitometrisi ile Katı Dokular İzole Hücre Cezalar Analizi

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

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 ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Skleraktin Hücre Popülasyonlarının İzolasyoniçin Floresan-Aktive Hücre Sıralaması

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

İskelet Kası Fibroblastlarından Eksozomların İzolasyonu ve Karakterizasyonu

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...