Biz USF Akım Sitometri Çekirdek Tesisi kabul ediyorsunuz. Biz kaynaklarının paylaşımı için Dr Denise Cooper teşekkür etmek istiyorum. Ayrıca kurma ve bu video filme kendi yardım için Maya Cohen, Laura Pendleton ve Diana Latour teşekkür etmek istiyorum. NN Doktora Bursu HRD # 0929435 NSF FG-LSAMP Köprüsü tarafından desteklenmektedir. Bu çalışma TG verilen Amerikan Kanser Derneği Kurumsal Araştırma Bursu # 93-032-13/Moffitt Kanser Merkezi tarafından finanse edildi.

Başlamadan önce, aşağıdaki çözümleri hazırlamak: % 3 Boyama Media (SM): 1X Phosphate Buffer Saline -3% Fetal Bovine Serum (FBS) (PBS) MACS Buffer (MB): - 1XPBS içinde Bovine Serum (BSA) 0.5% Albumin 1. Fare ile Hasat dalak <ol><li> (; TB tümör taşıyan) subkutan 1.5 x Panc02 10 5 fare hücrelerini 100 ul 1x PBS asılı olan yaş C57BL 6-8 hafta / 6 fareleri (Harlan) enjekte edilir. Kontrol fareler (naif) 100 ul 1XPBS alırsınız. </li><li> Yaklaşık 4 hafta sonra enjeksiyon, karbon dioksit asfiksisi ile farelerde euthanize. </li><li> Forseps ve makas kullanılarak künt diseksiyon ile farelerin Hasat dalak sonra denge ağırlıklarından yararlanılarak. Ayrı yerde dalak, 1 x PBS içeren 50 ml&#39;lik konik tüpler etiketli. </li></ol> 2. Tek hücreli Suspensio üretdalak gelen lökositler n Tüm işlemler bir Biyolojik Güvenlik Hood ve buz üzerinde tutulmalıdır hücreleri ve antikorların altında steril bir ortamda yapılmalıdır. <ol><li> Dibe doğru bardağı ağzına örgü ekran ekleyerek hücre ayrışma elek birleştirin. Sonra, oluklu tarafı dişli alana istinat halkası eklemek ve böylece yerinde ekranı tutan istinat bileziğini sıkmak için halka tuşunu kullanın. 10 ml 1 x PBS içeren Petri içinde monte elek yerleştirin. </li><li> Havuz dalak ve kullanım cam havaneli hücre ayrışma elek mesh ekrana ve Petri kabına dalak öğütmek için. Farelerin her tedavi grubu için tekrarlayın. </li><li> Bir 70 um hücre süzgecinden ve 5 ml serolojik pipet kullanılarak bir 50 ml konik tüpe Filtre hücre süspansiyonu. 5 dakika 12.000 rpm (250-300 xg) santrifüjleyin. </li><li> Dalak başına 5 ml 1 x RBC parçalama tamponunda süpernatan ve Pastör pipetiyle pelet Kaldır. Pipet yukarı ve aşağı şiddetle. 5 dk için oda sıcaklığında inkübe edilir. 1 x 20 ml PBS ilave edilerek reaksiyon durdur. Pipet yukarı ve aşağı şiddetle. 5 dakika 12.000 rpm (250-300 xg) santrifüjleyin. </li><li> 20 ml steril 1 x PBS süpernatan ve Pastör pipetiyle pelet çıkarın. Pipet yukarı ve aşağı şiddetle. </li><li> 50 ul 5x10 5-1x10 6 hücreleri (- 2x10 7 hücre / ml 1x10 7 hücre / ml) &#39;e denk olacak şekilde tripan mavi ve% 3 SM istenen konsantrasyonda bir hemasitometre ile Pastör pipetiyle kullanılarak hücreleri hesaplama. </li></ol> 3. Akım Sitometri kullanarak MDSC Hücre yüzey Boyama / İmmünofenotipleme <ol><li> (; Mac-1-FITC; Gr-1-APC ve DAPI No Leke, NS) kontrolü ve dengeleme kontroller için deneysel örnekleri ve tek bir leke için bir 96-kuyucuklu V alt levhanın kuyuları etiketlemek. </li><li> 96-kuyulu plakalı V-altlı in / kuyu kendi kuyucuklara splenositlerin 50 ul eşdeğer 5x10 5-1x10 6 hücreleri ekleyin. Santrifüj5 dakika 12.000 rpm (250-300 xg) plakası. </li><li> Buz üzerinde 1.5 ml mikrosantrifüj tüp,% 3 SM inceltilmiş Fare BD Fc Bloğu (Sıçan anti-fare CD16/32 monoklonal antikor) ve &quot;Master Mix&quot; (MM) hazırlayın. Bir başlangıç ​​noktası olarak, başına iyi, 50 ul son bir birim için% 3 SM 1 mikrogram Fc Blok kullanın. </li><li> Dikkatle hücre peletlerin kesinti olmadan bir atık konteyner veya lavabo üzerinde, üzerinde hızla geri ters plakası tarafından 96-kuyucuklu V-altlı plakanın her gelen süpernatan kaldırmak ve. </li><li> Vortex, kısaca 5 saniye Fc Blok MM santrifüj ve 96-V-alt plaka tüm peletlerine 50 ul ekleyin. Yavaşça kendi kuyularında örnekler bırakarak, aşağı yukarı pipetleme tarafından iyice karıştırınız. Buz üzerinde karanlıkta 15 dakika süreyle plaka inkübe. 5 dakika 12.000 rpm (250-300 xg) plakası santrifüjleyin. </li><li> Örnekleri Fc Blok ile inkübe ise, buz üzerinde bir 1.5ml mikrosantrifüj tüp içinde% 3 SM inceltilmiş florokrom-konjuge antikor &quot;Master Mix&quot; (MM) hazırlayın. Antikorlar titre edilmelidirboyama işlemleri için optimal seyreltme belirlemek için. Bir başlangıç ​​noktası olarak, iyi örnek başına 50 ul son hacim% 3 SM bir 1:25 Mac-1-FITC seyreltme ve Gr-1-APC 01:20 seyreltme birleştirir. </li><li> Daha önce tarif edildiği gibi dikkatli bir şekilde 96-kuyucuklu V-altlı her bir yanı ile süpernatan kaldırın. </li><li> Vortex, kısaca 5 saniye için floresan-konjuge antikor boyama MM santrifüj ve kontrol ve deney peletlerine 50 ul ekleyin. Hafifçe yukarı ve aşağı pipetleme ile iyice karıştırın. Tek-leke tazminatlar için, oyuk başına 50 ul son hacim% 3 SM yağmurluk-1-FITC ile 1:25 seyreltme, Gr-1-APC ve 75 ng / ml &#39;lik DAPI 01:20 seyreltme, ekleme kendi kuyucuklara. (Hayır Leke) iyi lekesiz için 50 ul% 3 SM ekleyin. İyice karıştırılır ve buz üzerinde koyu içinde 30 dakika süreyle 96-kuyulu plakalı V-altlı hücreleri inkübe edilir. </li><li> Etiket FACS borular (5 ml, 12 mm x 75 mm polistiren tabanı yuvarlak bir tüp) 96-kuyulu plakalı V-altlı her bir kuyucuğa karşılık. Her FACS üzere 200 ul% 3 SM eklemetüp. </li><li> 5 dakika 12.000 rpm (250-300 xg) plakası santrifüjleyin ve süpernatantlar çıkarın. Her pelet için 100 ul% 3 SM ekleyerek pelet yıkayın ve yavaşça yukarı ve aşağı pipetleme ile iyice karıştırın. 5 dakika 12.000 rpm (250-300 xg) plakası santrifüjleyin. Bir kez daha adım yıkayın tekrarlayın. </li><li> Daha önce tarif edildiği gibi dikkatli bir şekilde 96-kuyucuklu V-altlı her bir yanı ile süpernatan kaldırın. 100 ul% 3 SM her pelletini ve iyice karıştırın. </li><li> Bunların sırasıyla etiketli FACS tüpe 96-kuyucuklu V-altlı plakanın her 100 ul tekrar süspanse pelet transfer edin. </li><li> Flow sitometri analizi öncesinde, 75 ng / ml DAPI kontrol etmek ve deneysel örnekler ve DAPI tek bir leke kompanzasyon kontrolü ekleyin. </li><li> MDSC yüzdeleri flow sitometri veri toplama gerçekleştirin. Negatif (hiç leke kontrol) ve tek pozitif kontrolleri kullanarak tazminat gerçekleştirin. Böylece günlük ölçekte ileri (FSC) karşı tarafında dağılım (SSC) görüntüleyen bir nokta komplo kurmak ve lökositilgi tespit edilebilir. Enkaz ve en ileri ve yana yayılım ile topaklar hariç tüm lökositlerin büyük bir kapı çizin. Bu ana kapıdan, DAPI-(canlı) hücreleri üzerine SSC karşı DAPI ve kapı görüntüleyen yeni bir nokta arsa oluşturun. Bu yeni bağımlı nüfus seçin ve çift pozitif Mac-1 karşı Gr-1 ve kapı görüntüleyen bir nokta komplo oluşturmak (Mac-1 + Gr-1 +) MDSC. </li></ol> 4. Gr-1 + Lökosit Manyetik Zenginleştirme <ol><li> 5 dakika 12.000 rpm (250-300 xg) de uygun şekilde etiketlenmiş FACS tüpleri ve santrifüj içine tablet 1x10 7 kalan boyasız lökositlerin. </li><li> 1.5 ml mikrosantrifüj tüp Gr-1-PE antikor MM hazırlayın. 10 7 hücre için, numune başına, 50 ul MB Gr-1-PE antikor 1:10 seyreltme kullanın. Daha fazla hücre sayıları için, buna göre hacimleri büyütmek. Kısaca, 5 saniye süreyle santrifüj FACS tüpler lökositlerin eklemek ve 4 15 dakika ° C&#39;de karanlıkta inkübe edin. </li&gt; <li> 5 dakika 12.000 rpm (250-300 xg) de FACS tüpleri, santrifüj MB 2 ml ekleyin ve supernatant atın. </li><li> 1.5 ml mikrosantrifüj tüpü Anti-PE mikroboncuk MM hazırlayın. 10 7 hücre için, numune başına, 200 ul MB anti-PE mikroboncuk bir 1:4 dilüsyonu kullanın. Daha fazla hücre sayıları için, buna göre hacimleri büyütmek. Kısaca, 5 saniye süreyle santrifüj FACS tüpler lökositlerin eklemek ve 4 15 dakika ° C&#39;de karanlıkta inkübe edin. </li><li> 5 dakika 12.000 rpm (250-300 xg) de FACS tüpleri, santrifüj MB 2 ml ekleyin ve supernatant atın. SB 3 ml Pastör pipetiyle pelet. Yeni, etiketli 50 ml konik bir tüpe 70 mikron süzgeç süzülür. </li><li> Hazırlayın ve prime Otomatik MACS Pro Separator. Uygun çözümler ve boş atık şişe ile Dolum tüm şişeler, gerekirse. Enstrüman açın ve sıvı kapları ve başlatmasından sonra kolon (lar) ın durumunu incelemek. Bütün semboller yeşil renkte olmalıdır. Menüsünde, üst gelen &quot;Ayrılık&quot; ı seçinardından menü çubuğu alt menü çubuğundan &quot;Şimdi yıkayın&quot;. Astarlama işlemini başlatmak için &quot;Çalıştır&quot; ardından pop-up seçeneği &quot;yıkayın&quot; seçeneğini seçin. Çimlendirme süreci başarıyla tamamlandığında, cihaz o Durum menüsü altında &quot;Ayrılık için hazır&quot; olduğunu gösterecektir. </li><li> Satır B negatif fraksiyon toplama ve satır C. pozitif fraksiyon toplanması için 15 ml konik tüp için bir satır, 50 ml konik tüp manyetik etiketli hücreleri ile tüp boyutları ve yeri için uygun soğutulmuş tüpü rakı 50 ml konik tüp seçin </li><li> Örneklerinden duyarlı modda etiketli hedef hücrelerin pozitif seçimi için &quot;POSSEL_S&quot; hücre ayırma programı seçin. Manyetik etiketli hedef hücreler otomatları sütun üzerinde korunur; etiketsiz hücrelerin mıknatısın otomatik çekilmesi üzerine satır B&#39;de negatif fraksiyon toplama tüpüne bırakılır, etiketli hedef hücreler tüp satır C pozitif toplama tüpüne çıkacak raf. </li></ol>+ Zenginleştirilmiş Lökositler Post-Sıralama Analizi <ol><li> Gr-1 Recount + ve Gr-1 - tripan mavi ve hemasitometre kullanarak kesirler. % 3 boyama ortam içinde istenen konsantrasyonda Pastör pipetiyle hücreleri 50 ul 5x10 5-1x10 6 hücreleri (1x10 7 hücre / ml - 2x10 7 hücre / ml) &#39;e denk olacak şekilde ve buna uygun olarak etiketlenmiş FACS tüpler 50 ul transfer. </li><li> Örnek başına 50 ul&#39;lik bir son hacim% 3 SM bir 01:25 seyreltildiğinde, sadece Mac-1 bir MM hazırlayın. Hücreleri Leke ve akım sitometri analizi için Gr-PE, Mac-1 FITC ve DAPI tek leke tazminatlar hazırlamak. 200 ul% 3 SM ve DAPI canlılığı boya maddesi olarak daha önce tarif ekleyin. </li><li> Yüzdeler ve ayrıca pre ve post-autoMACS zenginleştirme MDSC yüzdeleri karşılaştırmak için - Gr-1 + ve Gr-1 belirlemek için akım sitometri veri toplama gerçekleştirin. </li></ol> 6. Temsilcisi Sonuçlar Burada r göstermek<html> MDSC (Şekil 1) sıralama sonraki FACS için toplandı, naif dalak gelen Gr-1 + lökositlerin autoMACS zenginleşmesi için epresentative sonuçlar. 1x10 6 naif lökositlerin önce autoMACS sıralama, BD LSRII alet kullanarak MDSC yüzdeleri tespit etmek Mac-1-FITC ve Gr-1-APC antikorları ile boyandı. 1x10 7 naif lökositlerin sonra bir autoMACS Pro Ayırıcı kullanarak Gr-1 + lökositlerin zenginleştirilmesi için anti-Gr-1-PE antikorlar ve PE-mikro ile boyandı. Post-autoMACS zenginleştirme, Gr-1 Gr-1 yüzdeleri + ve Gr-1 - Toplanan kesirler akım sitometri kullanılarak değerlendirildi. 1x10 6 Gr-1 + lökositleri akım sitometri ile tümör taşıyan lökositlerin toplanmış olmayan zenginleştirilmiş için naif lökositlerin toplanmış zenginleştirilmiş MDSC yüzdeleri, analiz etmek ve karşılaştırmak için Mac-1-FITC antikoru ile çıkarıldı ve boyandı. fig1.jpg &quot;/&gt; Sıralama MDSC FACS için naif Gr-1 + lökositlerin Şekil 1. AutoMACS zenginleştirme. Dalak pankreas tümörü taşıyan ve naif farelerin hasat ve tek hücre süspansiyonları içine işlendi. Naif lökosit yüzey Flow sitometri analizi öncesinde autoMACS zenginleştirme (A), Mac-1 ve Gr-1 floresan-konjuge antikor ile boyandı. Akış sitometrisi Gr-1 analizi + (B) ve Gr-1 - Gr-1-PE antikor ve anti-PE mikro-küreler ile boyanmış havuzlanmış saf leuckocytes gelen Gr-1 + hücrelerinin (C) fraksiyonlar post-autoMACS zenginleştirme. MDSC ve Gr-1 Flow sitometri analizi Gr-1 + yüzdeleri sonrası autoMACS zenginleştirme Tümör taşıyan farelere (E) olmayan zenginleştirilmiş toplanmış lökosit (naif fareler, n = 5 göre toplanmış naif lökosit (D) + hücre ; tümörü taşıyan farelere, n = 3). MDSC ve Gr-1 yüzdeleri temsilcisi kontur araziler ve histogramları ortasında korunaklı vardır.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHIP+limits+immunoregulatory+capacity+in+the+T-cell+compartment.">SHIP limits immunoregulatory capacity in the T-cell compartment.</a> Blood. 113, 2934-2944 (2009).</li><li>Mack, E., Neubauer, A., Brendel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA+yield+from+small+cell+populations+sorted+by+flow+cytometry+applying+different+isolation+procedures.">Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures.</a> Cytometry. A. 71, 404-409 (2007).</li><li>Strauss, L., Czystowska, M., Szajnik, M., Mandapathil, M., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+responses+of+human+regulatory+T+cells+(Treg)+and+effector+T+cells+to+rapamycin.">Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin.</a> PLoS One. 4, e5994 (2009).</li></ol>

Çıkar çatışması ilan etti.

Bu, çeşitli hayvan modellerinde farklı lenf dokuları için de geçerlidir MDSC popülasyonlarının işleme ve immunophentyping için ayrıntılı bir yöntemdir. Özellikle, autoMACS zenginleştirme splenositlerin 4 Gr-1 azalması, dalak ve lenf düğümleri 5 ila myeloid alt saflaştırılması, CD8 kemik iliği nötrofiller 14 izolasyonu ve saflaştırılması + dalak den T hücreleri dahil olmak üzere çeşitli lökosit izolasyonu için kullanılabilir ve lenf düğü...

<table border="1"><tbody><tr><td> REAKTİF </td><td> ŞİRKET </td><td> # KATALOG </td><td> YORUMLAR </td></tr><tr><td> 1X Fosfat Tamponlu Salin </td><td> Thermo Scientific Hyclone </td><td> SH30028.02 </td><td> Ca 2 + / Mg 2 + / Fenol Kırmızı-free </td></tr><tr><td> Bovine Serum (BSA) ile Albümin </td><td> Sigma-Aldrich </td><td> A7906 </td><td> BSA PBS içinde rahatsız edilmeden çözmek edelim; Steril Çevre </td></tr><tr><td> Fetal bovin serumu (FBS) </td><td> Thermo Scientific Hyclone </td><td> SV3001403HI </td><td> İnaktive Isı; Steril Çevre </td></tr><tr><td> Sıçan anti-fare monoklonal antikor CD16/32 (Fc Blok) </td><td> BD Biosciences </td><td> 553142 </td><td> Steril Ortam </td></tr><tr><td> Anti-fare CD11b (Mac-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> SterilÇevre </td></tr><tr><td> Anti-fare Ly6G (Gr-1) APC </td><td> eBiosciences </td><td> 17-5931 </td><td> Steril Ortam </td></tr><tr><td> Anti-fare Ly6G (Gr-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> Steril Ortam </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> Seri Seyreltme Steril Ortam </td></tr><tr><td> Hücre Ayrılma Elek </td><td> Sigma-Aldrich </td><td> CD1-1KT </td><td> Kullanmadan önce Otoklav </td></tr><tr><td> 70-mikron süzgeç </td><td> BD Biosciences </td><td> 352350 </td><td> Steril Ortam </td></tr><tr><td> 1X RBC Lizis Tamponu </td><td> eBiosciences </td><td> 00-4333-57 </td><td> Steril ortam; kullanımdan önce oda sıcaklığına ısınmaya </td></tr><tr><td> Petri </td><td> Fisher Scientific </td><td> 08-757-12 </td><td> Steril Ortam </td></tr><tr><td> 50ml conical tüpler </td><td> Thermo Scientific </td><td> 339652 </td><td> Steril Ortam </td></tr><tr><td> 5ml 12X75mm polistiren tabanı yuvarlak tüpler </td><td> BD Biosciences </td><td> 352054 </td><td> FACS tüpleri olarak da bilinen; Steril Çevre </td></tr><tr><td> 96-kuyucuklu plaklar V-altlı </td><td> Corning </td><td> 3897 </td><td> Steril Ortam </td></tr><tr><td> Tripan Mavi </td><td> Cellgro </td><td> 25-900-CI </td><td> Steril Ortam </td></tr><tr><td> PE mikroküreler </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> Steril Ortam </td></tr><tr><td> AutoMACS Pro Ayırıcı </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS Sütunlar </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> Tampon Koşu AutoMACS </td><td> Miltenyi Biotec </td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

MDSC olgunlaşmamış makrofajlar, dendritik hücreler ve paraziter enfeksiyon, inflamasyon, travmatik stres, Graft-versus-host hastalığı, diyabet ve kanser de dahil olmak üzere 1-7 patolojik durumlarda lenfoid organlarda birikir granülosit heterojen bir nüfus vardır. Farelerde, MDSC ekspres Mac-1 (CD11b) ve Gr-1 (Ly6G ve Ly6C) yüzey antijeni 7. Bu MDSC de önemli ölçüde genişletilmiş, çeşitli tümör taşıyan konaklar incelenmektedir dikkat ve 7-10 naif muadillerine göre anti-tümör immün yanıtı bastırmak için önemlidir. Ancak, patolojik durumuna bağlı olarak, bastırma 11,12 ile farklı mekanizmaları ve hedefleri ile MDSC farklı alt nüfusları vardır. Bu nedenle, canlı MDSC popülasyonlarının etkin bir yöntem izole etmek için in vitro ve in vivo bastırma onların farklı moleküler mekanizmalar durulaştırmada önemlidir. Son zamanlarda, Ghansah grubu, bir mürin pankreas kanseri modelinde MDSC genişlemesi rapor etmiştir. Bizim tümör taşıyan MDSC homeostazının kaybı görüntülemek ve naif MDSC 13 oranla baskılayıcı fonksiyonu artmıştır. MDSC yüzdeleri naif vs tümörü taşıyan farelerin lenf bölümlerinde önemli ölçüde azdır. Bu genellikle bu MDSC doğru karşılaştırmalı analizleri engellemektedir büyük bir uyarı vardır. Bu nedenle, önceden Floresans Aktif Hücre Sıralama (FACS) kadar naif farelerden Gr-1 + lökositleri zenginleştiren saflık, canlılığı artırır ve önemli ölçüde sıralama zamanı azaltır. Bu sıralama hızlı FACS için bolca bulunur Ancak, tümör taşıyan farelerin Gr-1 + lökositlerin zenginleştirme isteğe bağlıdır. Bu nedenle, bu protokol, biz MDSC işaretleyicidir ve zamanında MDSC sıralama için naif farelerin dalak gelen Gr-1 + lökositleri zenginleştiren son derece etkili bir yöntem tarif. İmmünokompetent C57BL / 6 fare mürin Panc02 ce ile inoküle edilmiştirLLS subkutan naif fareler 1XPBS alırsınız oysa. Yaklaşık 30 gün aşılama sonrası; dalak hücre ayrışma elek kullanarak tek hücre süspansiyonları içine hasat ve işlenir. Splenositlerin sonra Eritrosit (RBC) parçalanır ve bu lökositlerin bir kısım Akım Sitometri kullanarak immünfenotip MDSC yüzdeleri Mac-1 ve Gr-1 karşı florokrom-konjuge antikor kullanılarak boyandı edilir. Paralel bir deneyde, naif farelerin tüm lökositlerin otomatik Manyetik Aktif Hücre Sıralama (autoMACS) Pro Separator kullanarak PE-mikro inkübe ve olumlu seçilen floresan konjuge Gr-1 antikorları ile boyanan. Sonra, Gr-1 + lökositlerin bir kısım Akım Sitometri kullanarak MDSC oranlarda artış tanımlamak için Mac-1 antikorları ile boyanan. Şimdi, bu Gr1 + zenginleştirilmiş lökositler in vivo ve in vitro (naif vs tümör taşıyan) karşılaştırmalı analizlerde kullanılmak üzere MDSC sıralama FACS hazır </em&gt; deneyleri.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Bu myeloid türevi bastırıcı hücreler (MDSC) işaretleyicidir ve Gr-1 zenginleştirmek için hızlı ve kapsamlı bir yöntemdir<sup&gt; +</supFare dalak gelen&gt; lökositlerin. Bu yöntem uygulanabilir Gr-1 için zenginleştirmek için sıralama akım sitometri ve AutoMACS Hücre kullanır<sup&gt; +</supKullanım için MDSC sıralama FACS önce&gt; lökositlerin<em&gt; In vivo</em&gt; Ve<em&gt; In vitro</em&gt; Deneyleri.

Akım Sitometri ve Otomatik Manyetik Aktif Hücre Sıralama (AutoMACS) kullanarak Naive ve Pankreas Tümör taşıyan fareler ile myeloid türevi bastırıcı hücreler (MDSC) hazırlanması

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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Research

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Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

26.1K Görüntüleme.  University of South Florida Morsani College of Medicine. Bu myeloid türevi bastırıcı hücreler (MDSC) işaretleyicidir ve Gr-1 zenginleştirmek için hızlı ve kapsamlı bir yöntemdir + lökositlerin. Bu yöntem uygulanabilir Gr-1 için zenginleştirmek için sıralama akım sitometri ve AutoMACS Hücre kullanır + lökositlerin In vivo Ve In vitro Deneyleri.

Video: Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Sıralama Kök Hücre Tahliller ve Akış Primer Beyin Tümörü Doku İşleme

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Tıp

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

İnsan Serum içinde N-metil-D-aspartat reseptör ya da dopamin-2 reseptör antikorları algılamak üzere yüksek verimli Flow Cytometry hücre bazlı bir deneyde

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Nanobody ve Antikor Temelli Doğrulama<em&gt; İn Vivo</emFarelerde&gt; Tümör Xenograftlarında NIRF-görüntüleme Deneyler kullanma<em&gt; Ex vivo</em&gt; Sitometrisi ve Mikroskopi

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Çoklu-parametre floresans ile aktive edilen hücre sınıflandırma İnsan lenfatik endotel hücreleri İzolasyonu

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Moleküler Probe Optimizasyonu (a Fotosentetik Organizmaya Hücre Ölümlerini belirleme<em&gt; Microcystis aeruginosa</em&gt;) Akım Sitometri Kullanımı

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.