Biz değerli tavsiye için Nobuyuki Onai, Hitoshi Takizawa ve Markus Manz teşekkür ederiz. Bu çalışma İsviçre Ulusal Bilim Vakfı, İsviçre Kanser Ligi ve Fondazione Ticinese başına la RIcerca sul Cancro tarafından finanse edildi.

1. Kemik İliği gelen Hücre Süspansiyon hazırlanması <ol><li> Fare Euthanize ve karın ve geriye doğru olan paslanmaz pan ve sprey% 70 etanol içinde hayvan yerleştirin. Femur ve tibia arka bacak ve omurga toplayın. </li><li> Doğru sivri uçlu makasla ve bir gazlı bez ile bacak kemiklerinden omurga tüm yumuşak doku artıkları çıkarın. 50 uM β-merkaptoetanol, 100 uM MEM olmayan amino asitler ile desteklenmiş% 10 ısı ile inaktive edilmiş fetal bovin serumu (FBS), 1 mM sodyum piruvat MEM, 2 içeren RPMI medyumu ile 30 ml bir 50 mL Falcon tüp içinde kemiklerin deposunda mM L-glutamin, 100 ug / ml streptomisin ve 100 U / ml penisilin. </li><li> Steril filtrelerle önceden sterilize harç alt örtün. </li><li> Harç haline kemikleri aktarılır ve bir sürtünme yüzeyine monte etmek için bir filtre katmanı ile kapsamaktadır. </li><li> Falcon filtresi ile donatılmış bir 50 ml Falcon tüpü hazırlayın. </li><li> Kemikler parçalamakhomojen bir hücre süspansiyonu elde edilene kadar bir havaneli yardımıyla. </li><li> Tüpüne süspansiyon aktarın. </li><li> Kalan hücreler yıkayın ve hasat taze RPMI tam orta harç 5 ml ekleyin. </li><li> Homojenize kemikleri net görünmesine kadar son iki nokta tekrarlayın. </li><li> Geriye kalan kalıntı doku ortadan kaldırmak amacıyla hücre süspansiyonu transferi ve yeni bir tüp içinde filtre. </li><li> 5 dakika 1.500 rpm&#39;de santrifüj tüpü ve dikkatli aspirasyon ile süpernatantı kaldırın. Hücre pelletini rahatsız değil emin olun. </li></ol> 2. Fenotipik Analizi <ol><li> Yeniden süspanse ve PBS ile% 2 FBS 15 mL bireysel farelerin hücreleri yıkanır. </li><li> 5 dakika 1.500 rpm&#39;de santrifüj tüpü ve dikkatli aspirasyon ile süpernatantı kaldırın. Pelet rahatsız değil emin olun. </li><li> 5 ml PBS ile% 2 FBS ve sayım hücrelerinde pelet askıya. </li><li> Transferi 1 × 10 6 hücre / 96 kuyu yuvarlak altplakası. </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüje plakası. </li><li> Aşağıdaki yüzey işaretleyicileri karşı yöneltilmiş mAbs bir karışımı hazırlayın. Bunların seyreltilir olarak PBS ile gösterilir ve% 2 FBS hücreleri leke. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 PE-Cy5 1:200 ile konjuge </li><li> APC 1:100 c-Kit konjuge </li><li> Biotin 1:50 konjuge SCA1 </li><li> FITC 1:50 konjuge CD34 </li><li> PE 1:100 konjuge FcγR </li><li> PE-Cy7 1:100 IL-7R konjuge </li></ul></li><li> Süpernatant atın ve her kuyuya mAbs karması 50 uL ekleyin. Pipetleme tarafından tek hücrelere ayrışır. </li><li> 4 ° C&#39;de karanlıkta 20 dakika süreyle inkübe hücreleri </li><li> PBS 150 uL% 2 FBS ile iki kez yıkanır hücreleri. </li><li> 5 dakika 1.500 rpm&#39;de santrifüj plaka ve tersini plaka ile süpernatantı atın. </li><li> PBS içinde streptavidin seyreltilir ve% 2 FBS gibi hücrelerin leke belirtti. <ul type="disc"><li> Streptavidin conjuAPC-Cy7 1:300 için gated </li></ul></li><li> Her bir kuyucuğa solüsyonu 50 uL ekleyin. Pipetleme tarafından tek hücre süspansiyonu haline ayrışır. </li><li> 4 ° C&#39;de karanlıkta 10 dakika süreyle inkübe hücreleri </li><li> PBS 150 uL% 2 FBS ile iki kez yıkanır hücreleri. </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüje plakası ve süpernatant atılır. </li><li> PBS ile% 2 FBS 100 ul içinde tekrar süspansiyon hücreleri. </li><li> FACS numune edinin. </li></ol> 3. C-Kit + Hücrelerinin Manyetik Zenginleştirme <ol><li> PBS, pH 7.2, (BSA)% 0.5 sığır serum albümin, ve 2 mM EDTA ihtiva eden bir tampon çözeltisi hazırlayın. </li><li> APC ile konjuge c-Kit mAb ihtiva eden tampon çözeltisi 1 mL, en azından 10 fareden türetilmiş kemik kabak hücre pelletini 01:50 seyreltilmiştir. </li><li> 4 ° C&#39;de karanlıkta 20 dakika süreyle inkübe hücreleri </li><li> Tampon solüsyonu, 40 mL ile iki kez yıkanır hücreleri. </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüje tüpü ve dikkatli bir şekilde t kaldırmakdiye aspirasyon süpernatant. Pelet rahatsız değil emin olun. </li><li> Pastör pipetiyle hücre pelletini ve pelet doğrudan her ötanazi fare için Anti-APC mikroboncuk 50 ul ekleyin. </li><li> İyice karıştırılır ve 4 ° C&#39;de karanlıkta 20 dakika inkübe edilir ve </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüj tampon çözeltisi ve 40 mL eklenerek hücrelerin yıkanır. Tamamen süpernatant aspire. </li><li> Her üç fare ötanazi tampon çözelti 4 mL katları şeklinde Pastör pipetiyle hücre pelletini. </li><li> Uygun bir MACS Ayırıcı manyetik alanda Yer MACS LS sütunlar. Her üç fare ötanazi 1 sütun kullanın. </li><li> Tampon çözelti 4 mL ile durulama sütunlar hazırlayın. </li><li> Sütunlar üzerine hücre süspansiyonu uygulayın. </li><li> Geçmesine ve tampon çözelti 4 mL ile sütun yıkayın etiketsiz hücreleri toplayın. Tampon solüsyonu 4 mL üç kez ekleyerek yıkama adımları gerçekleştirmek. Kolon rezervuar boşaltılır yeni tampon ekleyin. </li><li> Ayırıcı ve Plac sütunları çıkarıne 15 ml falcon tüpleri üstündeki her biri. </li><li> Her sütun üzerine tampon solüsyonu 4 ml. Hemen sütuna pistonu iterek manyetik etiketli hücreleri temizler. </li><li> 5 dakika 1.500 rpm&#39;de santrifüj tüpleri ve dikkatli aspirasyon ile süpernatantı kaldırın. </li><li> Tek bir 15 ml Falcon tüp içinde tampon çözelti ve grup peletlerin 1 mL her pelletini. </li></ol> 4. Hematopoetik Kök Hücreler ve Lineage kararlı progenitörlerin Sıralama <ol><li> Aşağıdaki yüzey işaretleyicileri karşı yöneltilmiş mAbs bir karışımı hazırlayın. Bunların seyreltilir gibi hücreler leke 500 uL% 2 ve PBS FBS gösterilir. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 PE-Cy5 1:200 ile konjuge </li><li> APC 1:100 c-Kit konjuge </li><li> Biotin 1:50 Sca-1 konjuge </li><li> FITC 1:50 konjuge CD34 </li><li> PE 1:100 konjuge FcγR </li></ul></li><li> Previou gelen boru santrifüjes 5 dakika 1.500 rpm&#39;de bölümü ve dikkatli aspirasyon ile süpernatantı kaldırın. </li><li> Pelet için mAbs karışımı ekleyin. Pipetleme tarafından tek hücre süspansiyonu haline ayrışır. </li><li> 4 ° C&#39;de karanlıkta 20 dakika süreyle inkübe hücreleri </li><li> 14 ml PBS içinde% 2 FBS ile iki kez yıkanır hücreleri. </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüje tüpü ve süpernatant atılır. </li><li> Streptavidin ile seyreltilir olarak 500 uL PBS ile gösterilir ve% 2 FBS hücreleri leke. <ul type="disc"><li> APC-Cy7 1:300 konjuge Streptavidin </li></ul></li><li> Tüp çözüm ekleyin. Pipetleme tarafından tek hücre süspansiyonu haline ayrışır. </li><li> 4 ° C&#39;de karanlıkta 10 dakika süreyle inkübe hücreleri </li><li> 14 ml PBS ile% 2 FBS ile iki kez yıkanır hücreleri. </li><li> 5 dakika boyunca 1500 rpm&#39;de santrifüje tüpü ve süpernatant atılır. </li><li> PBS ile% 2 FBS 500 uL içinde tekrar süspansiyon hücreleri. </li><li> Eli için yeni bir 5 mL tüp içinde hücre süspansiyonu aktarılır ve filtresıralama prosedürü ile karışabilir hücresel agrega minate. </li><li> Sıralanır popülasyonlarının 7-AAD + ölü hücreleri çıkarmak amacıyla hücre süspansiyonu ila 7-Aad 5 uL (it 1:100 seyreltilmiş kullanım) ekleyin. </li><li> Aşağıdaki fenotipleri göre BD FACSAria ile Sıralama hücreleri: <ul type="disc"><li> LKS hücreleri: lin - c-Kit + Sca-1 + </li><li> LT-HKH&#39;ler LKS CD34 - </li><li> ST-HKH&#39;ler LKS CD34 + </li><li> GMP: lin - c-Kit + Sca-1 düşük FcγR yüksek CD34 + </li><li> AP üyeleri: lin - c-Kit + Sca-1 düşük FcγR düşük CD34 - </li><li> CMPS: lin - CD34 c-Kit + Sca-1 düşük FcγR düşük + </li></ul></li></ol> 5.. Temsilcisi Sonuçlar 1 gösterir ve elektronik perdeleme prosedür örnek ŞekilLin skoru yaygın lenfoid ataları (CLPs) - / c-Kit lo / interlökin (IL)-7R + hücreleri; Lin - / c-Kit + / Sca-1 + (LKS) ve Lin - / c-Kit + / Sca -1 lo (c-Kit +) hücreleri; c-Kit + kapısından, ortak miyeloid ataları (CMPS) FcγR lo CD34 + hücreler, FcγR hi CD34 + hücreleri ve / megakaryosit olarak granülosit / monosit ataları (GMP) olarak tanımlanır FcγR lo CD34 olarak eritrosit ataları (MEP) - hücreleri; LKS kapısı, LT-HSC ve ST-HSC gelen CD34 olarak tanımlanır - ve CD34 + hücreler, sırasıyla. Yukarıda açıklanan fenotipe göre CD34 ekspresyonunun hem de diğer ataları göre sıralanabilir LT-HSC ve ST-HSC; Bu kemik iliği hücre süspansiyonu manyetik bilye (Şekil 2) c-Kit + hücreleri için zenginleştirilmiş olabilir. HKH&#39;ler ana olabilir<html> hücreleri nükleotid bağlayıcı bileşik tedavi protokollerinin geliştirilebileceğini 11 gibi nükleer kırmızı (DRAQ5) ile boyandı - CD34 Şekil 4, gösterildiği gibi canlı görüntüleme konfokal mikroskopi lize. HKH&#39;lerin olarak, ancak GMP değil, tedavi protokollerinin geliştirilebileceğini pozitif veziküller sitoplazma 12 görünür. + LT-HSC maruz kalan purinerjik P2 reseptörlerinin aktivasyonu sitosolik Ca 2 artış gösterir Şekil 5, gösterildiği gibi Dahası, sıralanır HKH&#39;ler fonksiyonel deneylerde kullanılan adenosin trifosfat-(ATP) ve 12 ila ionomycin. <img alt="Şekil 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> Şekil 1. Lin-BM hücrelerinin Temsilcisi nokta arsa analizi. Protokol metninde belirtildiği gibi kemik iliği hücre süspansiyon hematopoietik öncü ve HKH&#39;lerin belirlemek ve skor akış sitometresi at Elektronik yolluk prosedürü boyandı. pload/3736/3736fig2.jpg &quot;/&gt; Şekil 2. Kemik iliğinden c-Kit + hücrelerin seçici zenginleştirme için bir planı. <img alt="Şekil 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> Şekil 3 elektronik kapılı LKS CD34 Hücre döngüsü analizi -. Sağlıklı kontrol ve Ki-67 antikoru ve DAPI ile boyanarak İBH olan farelerde HKH&#39;ler. Hücreler sabit ve / Lyse Fix ve 1 mcg / ml FITC Ki-67 konjuge ve DAPI ile boyanarak takip tamponları Perm ile permeabilize edildi. Bisiklete binme hücreler ancak LKS CD34 tüm saptanabilir eğer - sağlıklı kontrollerin 12 HKH&#39;ler bölmesi. <img alt="Şekil 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> . Nükleotid bağlayıcı bileşik tedavi protokollerinin geliştirilebileceğini ve nükleer kırmızı (DRAQ5) ile boyandı HKH&#39;ler ve GMP - Şekil 4 CD34 sıralanmış FACS görüntüleme konfokal mikroskopi yaşayın. Küçük kadran tedavi protokollerinin geliştirilebileceğini pos göstermekitive veziküller HKH&#39;ler sitoplazmasında algılandı, ancak GMP değil. <img alt="Şekil 5," src="/files/ftp_upload/3736/3736fig5.jpg" /> Şekil 5. Sitosolik Ca 2 + Fura-2 yüklü ve ATP ve ionomycin ile uyarılan sıralanmış HKH&#39;ler yükselme. Tek hücreden izleri gösterilmiştir. Tüm hücreleri + ATP ilavesinden sonra sitozolik Ca 2 belirgin artış gösteren ekstrasellüler ATP duyarlı idi.

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Çıkar çatışması ilan etti.

Burada anlatılan yöntem tek tek fareleri (Şekil 1) in hematopoiesis hızlı ve doğru analizine olanak tanır. Fare inflamasyon modelleri, otoimmünite, immün yetmezlik, dejeneratif hastalıklar, metabolik hastalıklar ve kanser de dahil olmak üzere çeşitli deneysel ayarları, bu analiz hematopoiezis patolojik durumun etkisini ele veriyor. Şekil 3 elektronik kapılı LKS üzerinde hücre bisiklet aktivitesi analizini gösterir CD34 - Ki-67 mab ve DAPI ile...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42401 </td></tr><tr><td> MEM NEAA 100X </td><td> Gibco </td><td> 11140 </td></tr><tr><td> Sodyum piruvat </td><td> Gibco </td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15070 </td></tr><tr><td> PBS </td><td> Gibco </td><td> 20012 </td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Hücre Süzgeç 40 mikron </td><td> BD Falcon </td><td> 352340 </td></tr><tr><td> 7-AAD Boyama çözüm </td><td> BD Pharmingen </td><td> 559925 </td></tr><tr><td> Tampon Lyse / Fix </td><td> BD Pharmingen </td><td> 558049 </td></tr><tr><td> Tampon III Perm </td><td> BD Pharmingen </td><td> 558050 </td> &lt;/ Tr&gt; <tr><td> Ki-67 </td><td> BD Pharmingen </td><td> 556026 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> CD4 (GK1.5) </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8 (53-6,7) </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3 (145-2C11) </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R (RA3-6B2) </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19 (6D5) </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> Gr1 (RB6-8C5) </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> Tre119 (TER-119) </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1 (PK136) </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> c-Kit (2B8) </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> Sca-1 (D7) </td><td> eBiosciğımız uydunun </td><td> 135981 </td></tr><tr><td> CD34 (RAM34) </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcγR (2.4G2) </td><td> eBioscience </td><td> 553145 </td></tr><tr><td> Anti-APC mikroküreler </td><td> Miltenyi Biotec </td><td> 130-090-855 </td></tr><tr><td> LS Sütunlar </td><td> Miltenyi Biotec </td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

Kemik iliği HKH&#39;ler ve daha olgun kan hücrelerinin soy ataları ikamet ve yetişkin bir organizmanın bir farklılaşmanın temel sitedir. HKH&#39;lerin ömür boyu 1 için tüm kan hücre soylarının yaratabilmeleridir pluripotent hücrelerin bir dakika hücre popülasyonu oluşturmaktadır. Kemik iliğindeki HKH&#39;ler homeostazının moleküler diseksiyonu hematopoezisi, onkoloji ve rejeneratif tıp açısından önemli olmaktadır. Biz floresan antikor ve bireysel farelerde hematopoetik progenitör alt ve HKH&#39;lerin dağılımı (Şekil 1) puan almak için akım sitometri içinde elektronik yolluk prosedürü ile etiketleme protokolü tanımlar. Ayrıca, yaygın hematopoietik öncü yanı sıra uzun vadeli (LT) ve c-Kit eksprese eden hücrelerin manyetik zenginleştirme tarafından toplanan kemik iliği hücre süspansiyonları gelen HKH&#39;ler yeniden yapılandırılması kısa vadeli (ST) zenginleştirmek için bir metodu tanımlar. Elde edilen hücre hazırlanması için in vitro olarak seçilen alt sıralamak ve etmek için kullanılabilirİn vivo çalışmalar işlevsel (Şekil 2). Hem trabeküler osteoblastlar 2,3 ve sinüzoidal endotel 4 kemik iliğinde HKH&#39;ler destekleyen fonksiyonel niş oluşturmaktadır. N-kaderin + osteoblastlar 3 alt ve ligand anjiyopoietin-1 5 ile HKH&#39;ler ifade reseptör tirozin kinaz Tie2 etkileşimi de dahil olmak üzere osteoblastik niş içinde çeşitli mekanizmalar HKH&#39;ler sessizliğini belirlenmesinde hemfikir. Kemik iliğinde &quot;Hazırda&quot; aşırı bisiklet aktivitesi üzerine 6 çoğaltma ve sonunda yorgunluktan HKH&#39;ler korumak için çok önemlidir. Böyle Toll-like reseptör ligandlar 7 ve interferon-α 6 gibi doğuştan gelen bağışıklık sistemi hücrelerinde etkili eksojen uyarılara da soyu adamış ataları içine yayılması ve HKH&#39;lerin ayrımı uyarabilir. Son zamanlarda, lin içinde uyuyan fare HKH&#39;ler nüfusu - c-Kit + Sca-1 + CD150 + CD48 - CD34 - nüfusu 8 tarif edilmiştir. Hücrelerin Sıralama burada açıklandığı gibi hematopoietik öncü zenginleştirilmiş hücre süspansiyonu ile CD34 ekspresyonunun dayalı sağlar sakin kendini yenileyen LT-HKH&#39;ler ve ST-HKH&#39;ler 9 hem de izolasyon. Soyu pozitif hücrelerinin tükenmesine ve CD48 ve Flk2 antikorları ile birlikte LT-KYH sıralama göre benzer bir prosedür, daha önce 10 tarif edilmiştir. Bu yazıda fenotipik karakterizasyonu ve farklı fizyolojik ve patolojik şartlarda hematopoez izlemek için yararlı olabilir hematopoietik öncü, ex vivo hücre döngüsü analizi için bir protokol sağlar. Ayrıca, biz faktörleri ve hücre biyolojisi ve sinyal iletimi deneyleri yanı sıra transplantasyon için kendi kendini yenileme, genişleme ve farklılaşma düzenleyen mekanizmaları tanımlamak için kullanılabilir HKH&#39;ler, prosedürü sıralama bir FACS açıklar.

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Sitometrisi kemik iliği hematopoietik öncü dağıtımı yanı sıra verimli yalıtmak için son derece saf hematopoetik kök hücreleri (HKH&#39;ler) analiz etmek için bir yöntem tarif edilir. İzolasyon ^ işleminde esas olarak hücresel ve moleküler çalışmalar için HKH&#39;ler arındırmak için sıralama c-Kit + hücreleri ve hücre manyetik zenginleştirme dayanmaktadır.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Research

JoVE Journal

Biology

18.6K Görüntüleme.  Bellinzona (Switzerland). Sitometrisi kemik iliği hematopoietik öncü dağıtımı yanı sıra verimli yalıtmak için son derece saf hematopoetik kök hücreleri (HKH'ler) analiz etmek için bir yöntem tarif edilir. İzolasyon ^ işleminde esas olarak hücresel ve moleküler çalışmalar için HKH'ler arındırmak için sıralama c-Kit + hücreleri ve hücre manyetik zenginleştirme dayanmaktadır.

Video: Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Biyoloji

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Murin Yolk Sac ve AGM Erken Hematopoetik Kök Hücre İzolasyonu

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral aracılı demonte sırasında Ex vivo İnsan Hematopoetik Kök Hücre Eritropoiesis

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Kas Uydu Hücreleri İzolasyon, Kültür ve Transplantasyon

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Murin Vana Endotel Hücreleri İzolasyon

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Kemirgen Embriyonik retroviral Enfeksiyon Hematopoetik Farklılaşma Analizi için Hücre Türetilmiş embriyoidlerin Vücut Kök Hücreler

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...