Biz flow sitometri cihazı ve sıralama hizmetleri sağlanan Yüksek Hızlı Hücre Sıralayıcısı Çekirdek, kullanmak için teşekkür Alvin J. Siteman Kanser Merkezi, Washington Üniversitesi Tıp ve St Louis, MO Barnes Yahudi Hastanesi. Bu çalışma Ulusal Sağlık Enstitüleri Sağlık Grants R01 HL082549 ve K02 HL084179 (NRY) tarafından desteklenmiştir. Siteman Kanser Merkezi, NCI Kanser Merkezi Destek Hibe P30 CA91842 tarafından kısmen destekleniyor.

1. REAKTİFLERİN HAZIRLANMASI <ol><li> Fms ilgili tirozin kinaz 3 steril 1000x stok çözelti hazırlayın (FLT-3) ligand, makrofaj / granülosit koloni-interlökin (IL) -3 uyarıcı faktör (GM-CSF), kök hücre faktörü (SCF), trombopoetin (TPO), ve IL-6, üreticinin talimatlarına göre. Steril Retronectin stok solüsyonu (1mg/ml) da üreticinin talimatlarına göre hazırlayın. Küçük alikotları bu stok çözümleri Böl ve tekrarlanan donma-çözülme önlemek için -20 ° C&#39;de tutmak. </li><li> IMDM içinde% 2 FBS hazırlayın. </li><li> % 20 FBS son konsantrasyonları, 2 mM Glutamin, 100 adet / ml Penisilin / Streptomisin (PS) ile tam IMDM orta hazırlayın. Kullanmadan önce aşağıdaki sitokinler: 100 ng / ml FLT-3 ligand, 20 ng / ml GM-CSF, 100 ng / ml SCF, 100 ng / ml TPO, 50 ng / ml, IL-3 ve 100 ng / ml IL-6. </li><li> HBSS% 1 BSA (fenol kırmızısı olmadan Ca ve Mg-) ve D-PBS (Ca ve Mg-serbest)% 2 BSA hazırlayın. 4 0.22 mikron şırınga filtreler ve mağaza ° C geçerek çözümler sterilize </li><li> 4 HBSS ve mağaza% 0.02 EDTA hazırlayın ° C. </li><li> Alikotları ilgi ve mağaza DNA -80 ° C kodlama galv pseudotyped retrovirüs hazırlayın Bu prosedür, bu makalenin kapsamı dışındadır, ancak başka yerlerde açıklandığı (1-3). </li></ol> Giemsa Boyama için <ol start="7"><li> Kullanmadan önce, temiz bir cam şişe 1 mikron filtre kağıdı ile iki kez Wright / Giemsa leke çözüm filtre. </li><li> Giordano formülü - Kullanımdan hemen önce, filtrelenmiş Wright / Giemsa leke çözüm ve fosfat tampon çözeltisi, pH 6.4 200 ml, 50 ml karıştırılarak, Wright, / Giemsa buffer pH 6.4 hazırlamak. </li><li> Fosfat tampon çözeltisi, pH 6.0, 3.5 LH 2 O. 27.3 g KH 2 PO 4 ve 4.62 g Na 2 HPO 4 eriterek hazırlayın </li><li> H 2 O.% 50 metanol hazırlayın </li></ol> 2. PROSEDÜRÜ Çözülme ve pre-harekete geçirmek CD34 + hücreler <ol><li> 37 CD34 + donmuş hücreleri hızla bir şişe çözülme ° C son küçük bir buz kristali kalana kadar hafifçe sallayarak hücre süspansiyonu (1 ml) ve 50 ml konik tüp transferi. Şişeden geri kalan hücreler yavaşça oda sıcaklığında 1 ml ile durulayın% 2 FBS / IMDM ve hafifçe dönen 50 ml tüp açılır akıllıca ekleyin. 3 dakika bekleyin. Sonra, yavaş yavaş hafifçe karıştırarak 2 ml% 2 FBS / IMDM ekleyin ve 3 dakika bekleyin. Son hacim 32 ml ulaşana kadar, FBS / IMDM seyreltilmiş hücre süspansiyonu olarak 3 dakika aralıklarla aynı hacimde% 2 ekleyerek eklemeler arasında yavaşça dönen aynı işlemi tekrarlayın. </li><li> Oda sıcaklığında 10 dakika boyunca 250 xg&#39;de santrifüjleyin ve yaklaşık 0.5 ml geride bırakarak süpernatant kaldırmak. </li><li> 20 ml% 2 FBS / IMDM askıya alınması ve oda sıcaklığında 8 dakika boyunca 200 xg&#39;de santrifüj pelet yıkayın. </li><li> Süpernatantı ve yaklaşık 0,5 x 10 6 hücre / ml tam bir IMDM ortamda hücrelerin askıya . Mikrotüp, karışımı içine hücre süspansiyonu aynı Tripan mavisi çözeltisinin hacmi (% 0.4) ile 10 ul alın ve bir hemasitometre kullanarak saymak. </li><li> (1.3) sitokinler ile desteklenmiş IMDM orta ekleyerek 0,1-0,15 X 10 6 hücre / ml hücreleri sulandırınız </li><li> Kültür, 2 gün boyunca% 5 CO 2 ile nemlendirilmiş bir 37 ° C inkübatör hücreler . </li></ol> Virüs ön yükleme retroviral transdüksiyon <ol start="7"><li> Iletimi gün, hazırlıklı olmak kuyuların sayısını belirlemek için ön yükleme virüs başlamadan önce hücreleri saymak. </li><li> Retronectin stok çözeltinin 25 mcg / ml ve 24-iyi arıtılmamış doku kültürü plakası her bir kuyunun 400 ul ekleyin. Ön kat laminer akış biyogüvenlik kaput 2 saat oda sıcaklığında inkübe edin. </li><li> Retronectin çözüm çıkarın ve her bir kuyu için D-PBS içinde% 2 BSA 400 ul ekleyin. Oda sıcaklığında 30 dakika inkübe edin. </li><li> Virüs stok solüsyonları çözülme ve buz üzerinde tutmak. BSA çözüm çıkarın, her iyi 0.5 ml virüs hazırlık ve 4 2200 xg&#39;de santrifüj ° C de 15 dakika boyunca. </li><li> Kuyulardan virüs çözümü çıkarın ve 2.10 en fazla üç defa açıklanan virüs yükleme tekrarlayın. </li><li> Soğuk 0.5 ml HBSS (+ Ca, Mg) veya IMDM her iyice durulayın. </li><li> Oda sıcaklığında 8 dakika boyunca 200 xg&#39;de santrifüj önceden aktive edilmiş CD34 + hücreler toplayın. </li><li> Sitokinler ile tam IMDM orta 1.5 ml başına 0,1-0,15 x 10 6 hücre hücrelerin tekrar </li><li> Her iyi 1.5 ml hücre süspansiyonu ekleyin ve 2 gün için% 5 CO 2 ile nemlendirilmiş bir 37 ° C inkübatör inkübe edin. </li></ol> Hücre sıralama ve toplama hücreleri transduced <ol start="16"><li> Yavaşça ama iyice yukarıda virüs yüklü plaka hücreleri askıyakonik bir tüp içine 50 mikron CellTrics hücre filtre ile filtre. Tripan mavisi çözeltisinin eşit hacmi olan bir mikrotüp, karışımı içine atın ve 10 ul hücre süspansiyonu bir hemasitometre kullanarak saymak. 0.8 ml soğuk% 0.02 EDTA / HBSS (fenol kırmızısı olmadan Ca ve Mg-free) her iyice durulayın ve 50 mikron CellTrics hücre filtre ile aynı toplama tüpüne ekleyin. </li><li> 8 dakika 4 ° C 200 xg&#39;de hücreler santrifüj, ve 4, 8 dakika için 200 xg&#39;de santrifüj tarafından soğuk HBSS ile pelet (fenol kırmızısı olmadan Ca ve Mg-free) yıkamak ° C </li><li> BSA / HBSS (fenol kırmızısı olmadan Ca ve Mg-) 15 X 10 6 / ml veya hücre sıralama için 0.250 ml minimum hacim konsantrasyonu% 1 pelletini tekrar. Hücreler karanlıkta buz üzerinde tutun. Soğuk% 20 FBS / IMDM / sıralanmış hücreleri toplamak için Glutamin / PS orta içeren bir tüp hazırlayın. Washington Üniversitesi Tıp MoFlo yüksek hızda sıralayıcı (Dako, Glostrup, Danimarka) kullanarak Fakültesi Alvin J. Siteman Kanser Merkezi Yüksek Hızlı Hücre Sıralayıcısı Çekirdeği sıralama yapılır. </li></ol> CFC testi <ol start="19"><li> CFC tahlil medya 3 ml alikotları gerekli sayıda çözülme. Vortex kuvvetlice karıştırın ve tüpler kabarcıkları eklemeden önce hücrelerin yüzeye çıkması için mevcut en az 5 dakika bekletin. </li><li> Sıralanmış her bir örnek için hücre konsantrasyonu elde etmek için, hücre sayısı yaklaşık hücre süspansiyonu hacmi sıralama tesisi tarafından sağlanan bölün. 3.000 atın virüs transduced, soğuk% 2 FBS / IMDM içeren steril bir mikrotüp ayrılır hücreleri. Ön ayarlamak son süspansiyon hacmi yaklaşık 0.3 ml olacak FBS / IMDM% 2 birim. </li><li> Hücreleri, askıya alma ve 2 mM L-glutamin% 1 metilselüloz oluşur Methocult GF + H4435,% 30 FBS,% 1 BSA, 10 -4 M 2-mercaptoethanol, 3 ml kısım için tüm 0.3 ml hücre süspansiyonu transfer 50 ng / ml SCF, 20 ng / ml, GM-CSF, 20 ng / ml, IL-3, 20 ng / ml, IL-6, 20 ng / ml G-CSF ve 3 adet / ml eritropoietin IMDM. % 1.3 metilselüloz oluşur İnsan Metilselüloz Zenginleştirilmiş Medya (Ar-Ge Sistemleri),% 25 FBS,% 1 BSA, 5 x 10 -5 M 2-mercaptoethanol, 2 mM L-glutamin, 50 ng / ml SCF, 20 ng / 20 ml GM-CSF, IL-3 ng / ml, 20 ng / ml, IL-6, 20 ng / ml G-CSF ve 3 adet / ml eritropoietin IMDM de kullanılıyor olabilir. </li><li> Vortex şiddetle karışımı tam olarak yükselişi ve 3-4 kez düşme. Tüp 3 dakika bekletin. </li><li> 3 ml şırınga göstergesi, 16 künt uç iğne takın ve 2.2 ml kadar çizin. Büyük kabarcıklar yukarı çekmek etmeyin; başında birkaç kez basarak onları kovmak. Her iki adet 30 mm arıtılmamış çanak içine 1.1 ml çevirerek dışarı itin ve karışımı eşit olarak yaymak. </li><li> 3 ml steril su içeren bir su çanağı ile birlikte 100 mm plakası yinelenen bir tabak koyun. 14 - 17 gün için Kültür. </li><li> Göre koloniler karakterize bir skor ızgara ile işaretlenmiş bir kültür çanak 40x büyütme ters bir mikroskop ile morfoloji ve skor. Saf eritroid, myelomonositik ve karışık: testin amaçları için, koloniler 3 kategoride sınıflandırılır. Daha fazla koloni subclassification için üreticinin talimatlarına bakın. </li><li> Tüm CFC tahlil plaka büyütme olmaksızın düzenli bir 600 dpi tarayıcı ve kolonilerin fotomikrografı renkli bir kamera ile donatılmış bir inverted mikroskop kullanılarak alınabilir düşük-güç (40x) ile taranabilir. </li><li> Tüm CFC tahlil plaka hücre farklılaşması ve proliferasyonu daha fazla analiz için, oda sıcaklığında% 2 FBS / IMDM birkaç cilt olarak askıya tarafından kurtarıldı. 10 dakika 4 ° C için 300 xg&#39;de santrifüj sonra, hücrelerin% 2 FBS / IMDM, sayılır ve her iki akım sitometri için antikorlar ile boyanarak ya da Giemsa boyama için sitospin santrifüj kullanarak slayt transfer yeniden süspanse. </li></ol> Antikor boyama ve flow sitometri <ol start="28"><li> 4 8 dk boyunca 200 xg&#39;de Santrifüj hücreler HBSS soğuk% 50 normal fare serumu ° C ve tekrar süspansiyon (fenol kırmızısı olmadan Ca ve Mg-ücretsiz). </li><li> Yaklaşık 5 x 10 5 hücre tüp başına 80 ul (12 x 75 mm yuvarlak alt tüpler) koyun ve buz üzerinde tutmak. Antikor karışımı (yaklaşık 20 ul-antikor kontrolleri için karışım veya negatif kontrol solüsyonu) ve tüpler hafifçe dokunarak karışımı ekleyin. Herhangi bir antikor ya da her florokrom için bir anti-CD45 antikoru ile kalibrasyon kontrolü tüpleri hazırlayın. Tüpler, 20 dakika boyunca karanlık ve inkübe buz koyun. İlk 10 dakika sonra tüpleri hafifçe dokunarak karıştırın. 20 dakika inkübasyondan sonra, soğuk HBSS tüpleri doldurmak ve 4, 8 dk boyunca 200 xg&#39;de santrifüj ° C. Soğuk% 0.5 paraformaldehid / HBSS pelletini tekrar. Antikor aşağıdaki karışımlar genelde analizi için kullanılır. Miyeloid farklılaşma: CD11b (fikoeritrin-konjuge klon ICRF44) / CD33 (allophycocyanin konjuge klon WM53) / CD45 (fikoeritrin-Cy7-congjugated klon HI30). Eritroid farklılaşması için: CD71 (fikoeritrin-konjuge klon M-A712) / CD235a (allophycocyanin-konjuge klon GA-R2) / CD45 (fikoeritrin-Cy7-congjugated klon HI30). </li><li> Flow sitometri, Alvin J. Washington Üniversitesi Tıp Okulu&#39;nda Siteman Kanser Merkezi, 5 renk ve iki lazerler (BD Biosciences) yükseltilmiş bir FACScan akış sitometresinde Yüksek Hız Hücre Sıralayıcısı Çekirdek ve CellQuest (BD Biosciences) veya kullanılarak analiz FlowJO v7.2.4 (Tree Star, Inc, Ashland, OR, ABD) yazılımı. </li></ol> Giemsa boyama <ol start="31"><li> CFC tahlil plakaları kurtarıldı yaklaşık 30.000 hücreleri 0.3 askıya yukarıda açıklanan ml% 2 FBS / IMDM ve oda sıcaklığında 10 dakika 1.000 rpm&#39;de sitospin santrifüj kullanarak slayt aktarılır. </li><li> Aşağıdaki sırayla çözümler içeren dokuz boyama damarları ayarlayın. <ol><li> Mutlak Metanol </li><li> Wright / Giemsa stok solüsyonu </li><li> Wright / Giemsa tamponu, pH 6.4 </li><li> % 50 H 2 O Metanol </li><li> H 2 O </li><li> H 2 O </li><li> Fosfat tamponu, pH 6.0 </li><li> H 2 O </li><li> H 2 O </li></ol></li><li> Slaytları slayt taşıyıcı içine yerleştirin ve 2 dakika için mutlak metanol daldırma (# 1) ve fazla metanol kurulayın. </li><li> Hemen 5 dakika Wright / Giemsa stok solüsyonu slayt taşıyıcı (# 2) batırın. </li><li> Wright / Giemsa tamponu, pH 6.4 (# 3) taşıyıcı aktarın ve 10 dakika inkübe edin. </li><li> (# 4) iki kez% 50 metanol taşıyıcı Daldırma, H 2 O (# 5), H 2 O (# 6) başka bir 10 kat, 10 kat ve daha sonra 5 kez Fosfat tamponu, pH 6.0 (# 7) . </li><li> H 2 O (# 8) taşıyıcı transferi ve 2 dakika boyunca inkübe edin. H 2 O (# 9) yıkama işlemi tekrarlayın. </li><li> Slaytlar taşıyıcı tamamen kurumaya bırakın. Her bir slayt çıkarın ve lekeleri çıkarmak için, metanol-batırılmış Kimwipes slayt geri silin. Cytoseal 60 bir damla ve mühür cam bir kapakla yerleştirin. </li><li> Olympus BX51 mikroskop kullanılarak her Giemsa lekeli slayt için 500 hücre diferansiyel sayımı yapılır. Hücreler beş kategoriye ayrılır: ilkel hücrelerden patlamalar ve promyelocytes; ara miyeloid hücrelerin myelocytes ve metamyelocytes; olgun miyeloid hücrelerin bantları, segmente nötrofiller, monositler ve makrofajlar; ara eritroid hücreler ara hemoglobinization sahip hücreler içerir; ve olgun eritroid hücreler içerir tam hemoglobinization sahip hücreler. Fotomikrografı 60x petrol hedefi ile Olympus DP71 kamera ile alınır. </li></ol> 3.0) TEMSİLCİSİ SONUÇLAR: <ol><li> CFC tahlil: Bazı giriş hücre popülasyonu, prolifere farklılaştırmak ve 14-17 günlük kuluçka döneminde yarı-katı orta form koloniler. Bu deneyde, NUP98 HOXA9 onkogen ekspresyonu belirgin kırmızı (eritroid koloni) (Şekil 1) (3) oluşumuna neden olduğu açıktır. </li><li> Antikor Boyama ve Akış Sitometri: Akış sitometrik işaretleyicidir CFC tahlil plakaları toplanan hücreler, hücre nüfusun farklılaşması durumu hakkında bilgi sağlar . Belirli bir farklılaşma işaretleri karşı antikor kombine kullanımı, hücrelerin olgunlaşma soy ve derecesi tespit edilebilir. Bu durumda CD235a eritroid hücreleri tanımlamak için kullanılır oldu. Şekil 2A gösterildiği gibi, NUP98-HOXA9 CD235a + eritroid hücrelerin yüzdesi bir artışa neden olmuştur, ancak bu hücreler tarafından CD235a ifade parlaklık eritroid olgunlaşma inhibisyonu gösteren, kontrollere göre azaldığı gözlendi. Şekil 2B, miyeloid hücrelerin, CD33 pozitifliği sayesinde tespit edilir. NUP98 HOXA9 CD11b ifade parlaklık miyeloid olgunlaşma inhibisyonu (3) ile tutarlı bir azalma ile CD33 + hücrelerin sayısında bir azalmaya neden oldu. Böylece akış sitometri sonuçları NUP98-HOXA9 miyeloid ve eritroid hem olgunlaşma inhibisyonu ile eritroid hiperplazi neden olduğunu göstermektedir. </li><li> Giemsa Boyama: CFC tahlil plakaları toplanan hücreler morfolojik incelemesinde hücre popülasyonunun olgunlaşma soy ve derecesi hakkında bilgi sağlar. Morfolojik olarak elde edilen sonuçlar, akım sitometrik çalışmaları ile elde edilen farklı olabilir ve bu nedenle bu yöntemleri (3) birbirini tamamlayıcı niteliktedir. Deney en az 3 kez tekrarlanır ve her seferinde 500 hücre diferansiyel sayısı, yukarıda açıklandığı gibi hücre 5 kategoriye ayırmak için yapılır. Şekil 3&#39;te bu hücrelerin örnekleri dikkat çekti. Sonuçları arasında istatistiksel olarak anlamlı farklılıklar onkogen transduced örnek ve kontrolü olup olmadığını belirlemek amacıyla tablo ve analiz edilir. Bu durumda, sonuçlar NUP98-HOXA9 sayısında genel bir artışa neden olduğunu gösterdieritroid hiperplazi ve eritroid ve myeloid olgunlaşma (3) (gösterilmemiştir) inhibisyonu ile hücre. </li></ol> Şekil açıklamaları <img src="/files/ftp_upload/2195/2195fig1.jpg" alt="Şekil 1" /> Şekil 1: insan CFC morfolojisi NUP98-HOXA9 etkiler. İlköğretim insan CD34 + hücre retrovirally kontrolü MSCV IRES-GFP vektör veya vektör NUP98-HOXA9 ifade transduced, ve hücreler, GFP pozitifliği için sıralanır. CFC tayini için iki yinelenen plakaların her birine bin hücre numaralı seribaşı ve deney 3 4 bağımsız kez tekrarlandı. Büyütme (solda) ve düşük güç temsilcisi eritroid kolonilerin (sağda) fotomikrografı olmadan Temsilcisi levhalar (3) gösterilmiştir. <img src="/files/ftp_upload/2195/2195fig2.jpg" alt="Şekil 2" /> Şekil 2: Flow sitometri NUP98-HOXA9 tarafından bozulması insan birincil CD34 + hücre farklılaşması gösterir. (A) CD45 ve CD235a eritroid farklılaşması için akış sitometri: CFC plakalardan Hücreler (bkz. Şekil 1) antikorları ile hasat ve boyandı. (B) CD235a + kapı kontrol hücrelerine göre CD235a ifade göstermek için histogram (sağ panel) çizilen oldu miyeloid farklılaşma Flow sitometri: CFC plakalardan hücreler CD45 ve CD33 ile hasat ve boyandı. CD33 + kapı kontrole göre (sağ panel) CD11b ifade göstermek için bir histogram üzerinde çizilen oldu. Her bir geçidin giren hücrelerinin yüzdeleri gösterilmiştir (3). <img src="/files/ftp_upload/2195/2195fig3.jpg" alt="Şekil 3" /> Şekil 3: Hücre morfolojisi HOXA9 NUP98-farklılaşma bozulması vektör kontrol ile karşılaştırıldığında gösterir. Sitospin smear CFC plakalardan hazırlanan ve Giemsa ile boyandı. Fotomikrografı 60x petrol hedefi ile temsili alanlar alınmıştır. B: patlama; MM: olgun miyeloid, IM: orta miyeloid, ME: olgun eritroid, IE: Ara eritroid (3).

<ol>
	<li>Takeda, A., Goolsby, C., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUP98-HOXA9+induces+long-term+proliferation+and+blocks+differentiation+of+primary+human+CD34++hematopoietic+cells.">NUP98-HOXA9 induces long-term proliferation and blocks differentiation of primary human CD34+ hematopoietic cells.</a> Cancer Res. , 66-6628 (2006).</li><li>Yassin, E. R., Abdul-Nabi, A. M., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+NUP98-DDX10+oncogene+on+primary+human+CD34++cells:+role+of+a+conserved+helicase+motif.">Effects of the NUP98-DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif.</a> Leukemia. , (2010).</li><li>Yassin, E. R., Sarma, N. J., Abdul-Nabi, A. M., Dombrowski, J., Han, Y., Takeda, A., Yaseen, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+transformation+of+primary+human+hematopoietic+cells+by+the+oncogene+NUP98-HOXA9.">Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.</a> PLoS One. 4, e6719-e6719 (2009).</li><li>Nissen-Druey, C., Tichelli, A., Meyer-Monard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hematopoietic+colonies+in+health+and+disease.">Human hematopoietic colonies in health and disease.</a> Acta Haematol. 113, 5-96 (2005).</li><li>Pereira, C., Clarke, E., Damen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+colony-forming+cell+assays.">Hematopoietic colony-forming cell assays.</a> Methods Mol Biol. 407, 177-208 (2007).</li><li>Coulombel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hematopoietic+stem/progenitor+cells:+strength+and+drawbacks+of+functional+assays.">Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays.</a> Oncogene. 23, 7210-7222 (2004).</li><li>Hogge, D. E., Lansdorp, P. M., Reid, D., Gerhard, B., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection,+maintenance,+and+differentiation+of+primitive+human+hematopoietic+cells+in+cultures+containing+murine+fibroblasts+engineered+to+produce+human+steel+factor,+interleukin-3,+and+granulocyte+colony-stimulating+factor.">Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.</a> Blood. 88, 3765-3773 (1996).</li></ol>

CFC tahlil hematopoetik atalarıdır çoğalmasını ve farklılaşmasını modellerini belirlemek ve onkogenler (4, 5) etkilerini incelemek için yaygın olarak kullanılmaktadır. Bu koloniler tek bir progenitör soyu temsil eden ve ileri analiz için ayrı ayrı kaldırılabilir klonal bir tahlil, sıvı kültürler üzerinde bir avantaja sahiptir. Bu tür hücreler kullanılarak tespit edilir, uzun vadeli bir kültür başlatan hücre (LTC-IC) assay (6, 7). CFC testinin sınırlama, daha olgunlaşmamış atalarıdı...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>12440</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>06150</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin (PS)</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>FLT-3 ligand</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-19</td>
<td>&nbsp;</td>
<tr>
<td>GM-CSF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-03</td>
<td>&nbsp;</td>
<tr>
<td>SCF</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-07</td>
<td>&nbsp;</td>
<tr>
<td>TPO</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>300-18</td>
<td>&nbsp;</td>
<tr>
<td>IL3</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-03</td>
<td>&nbsp;</td>
<tr>
<td>IL6</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-06</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma- Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP118</td>
<td>&nbsp;</td>
<tr>
<td>Retronectin</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>T100A</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Life Technologies</td>
<td>14200</td>
<td>&nbsp;</td>
<tr>
<td>Trypan Blue solution (0.4%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
<tr>
<td>Methocult GF+ H4435</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>04445</td>
<td>&nbsp;</td>
<tr>
<td>Human Methylcellulose Enriched Media</td>
<td>&nbsp;</td>
<td>R &amp; D Systems</td>
<td>HSC005</td>
<td>&nbsp;</td>
<tr>
<td>Wright/ Giemsa stain</td>
<td>&nbsp;</td>
<td>Harleco</td>
<td>64571</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Solution, pH 6.4 - Giordano formula</td>
<td>&nbsp;</td>
<td>Ricca Chemical Company</td>
<td>1450</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A412-4</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 60</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>8310</td>
<td>&nbsp;</td>
<tr>
<td>Normal Mouse Serum </td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals</td>
<td>D208</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD11b phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555388</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD33 allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551378</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD71 phycoerythrin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555537</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD235a allophycocyanin-conjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>551336</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Human CD45 phycoerythrin-Cy7-congjugated</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>557748</td>
<td>&nbsp;</td>
<tr>
<td>24-well non-treated tissue culture plates</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-1147</td>
<td>&nbsp;</td>
<tr>
<td>30 mm non-treated dish</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27150 </td>
<td>&nbsp;</td>
<tr>
<td>100 mm tissue culture dish </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>27500</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2097</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>35-2070</td>
<td>&nbsp;</td>
<tr>
<td>Syringes 3 ml </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28240</td>
<td>&nbsp;</td>
<tr>
<td>16 gauge blunt-end, 1&frac12; inch needle </td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>28110</td>
<td>&nbsp;</td>
<tr>
<td>50 &mu;m CellTrics cell filter</td>
<td>&nbsp;</td>
<td>Partec</td>
<td>04-004-2327</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>0267110</td>
<td>&nbsp;</td>
<tr>
<td>TPX sample chambers</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>A78710018</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Superfrost/Plus Microscope Slides, Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>Shandon filter cards</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>5991022</td>
<td>&nbsp;</td>
<tr>
<td>Shandon cytospin slide holder</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>59920063</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Complete Staining Assembly 100</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>100</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark Corporation</td>
<td>34155</td>
<td>&nbsp;</td>
<tr>
<td>1 &mu;m filter paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28307-134</td>
<td>&nbsp;</td>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>Diaphot</td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>DS-F11</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX51 </td>
<td>&nbsp;</td>
<tr>
<td>Microscope camera</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DP71</td>
<td>&nbsp;</td>
<tr>
<td>Scanner</td>
<td>&nbsp;</td>
<td>Microtek</td>
<td>Scanmaker 4</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-812</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture incubator</td>
<td>&nbsp;</td>
<td>Sanyo</td>
<td>MCO-18AIC</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>&nbsp;</td>
<td>Shandon</td>
<td>Cytospin 2</td>
<td>&nbsp;</td>
<tr>
<td>Bench-top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5810-R</td>
<td>&nbsp;</td>
<tr>
<td>Water purification system</td>
<td>&nbsp;</td>
<td>Barnstead</td>
<td>Nanopure-Diamond</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

colony forming cell (cfc) assay for human hematopoietic cells

İnsan hematopoietik kök / progenitör hücreleri genellikle kemik iliği, kordon kanı veya periferik kandan elde edilen ve hemopoeziste ve leukemogenesis incelemek için kullanılır. Bunlar lenfoid ve myeloid soyları ayırt etmek için kapasite var. Koloni oluşturan hücre (CFC) tayini, yarı katı bir ortamda koloniler oluşturmak için yeteneklerini proliferasyon ve farklılaşma desen hematopoetik atalarıdır çalışma kullanılır. Sabit bir sayıda giriş hücrelerinin oluşturduğu kolonilerin sayısı ve morfolojisi ayırt etmek atalarıdır yeteneği hakkında ön bilgi vermek ve çoğalırlar. Hücreler bireysel koloniler veya bütün plaka numaraları ve akım sitometri ve Giemsa lekeli dia morfolojik değerlendirme kullanarak farklılaşma devletler daha da değerlendirmek için hasat edilebilir. Bu testte, ama lenfoid değil miyeloid farklılaşma değerlendirmek için yararlıdır. Bu bağlamda vadeli miyeloid granülositik, monositik, eritroid ve megakaryocytic soy kapsayacak şekilde daha geniş anlamda kullanılır. Biz periferik kandan elde edilen birincil insan CD34 + hücre farklılaşması onkogenler etkilerini değerlendirmek için bu testte kullandık. Bu amaçla hücreler için NUP98-HOXA9 Bu durumda, kontrol retroviral inşa veya ilgi onkogen ifade bir yapı ile transduced. Biz, ilgi ve GFP işaretleyici gen üreten bir bicistronic mRNA ifade yaygın olarak kullanılan bir retroviral vektör, MSCV IRES-GFP, istihdam etmektedir. Hücreler sitokinlerin varlığında retroviral transdüksiyon öncesinde iki gün boyunca büyüyerek önceden aktive edilir. Bir iki gün sonra, floresan ile aktive olan hücre sıralama (FACS) GFP + hücreler izole edilir ve sitokinler ile desteklenmiş ve koloniler genellikle yüzey, 14 gün kadar inkübe metilselüloz içeren yarı katı orta ile karışık. Kolonilerin sayısı ve morfolojisi belgelenmiştir. Hücreler daha sonra plakalar kaldırılır yıkanmış sayılır ve akım sitometri ve morfolojik inceleme tabi. Flow sitometri hematopoez sırasında ifade edilen hücre yüzey belirteçleri için özel antikorlar ile soy ve olgunlaşma aşamasında hakkında bilgi sağlar. Wright-Giemsa boyama sonrası mikroskop altında tek tek hücrelerin morfolojik çalışmalar, soy ve olgunlaşma ile ilgili daha fazla bilgi sağlar. Hücrelerin karşılaştırılması bir onkogen hematopoetik farklılaşma onkogen etkileri açığa transduced bu boş vektör kontrolü ile transduced.

Watch this Scientific Journal Video about Colony Forming Cell CFC Assay for Human Hematopoietic Cells at JoVE.com

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Koloni oluşturan hücre (CFC) tayini hematopoetik atalarıdır yarı-katı ortamda koloniler oluşturur in vitro testtir. Koloni morfolojisi, hücre morfolojisi ve akış sitometri bir arada, farklı hematopoetik soy boyunca çoğalırlar ve ayırt atalarıdır yeteneklerini değerlendirmek için kullanılır.

Koloni Oluşturan İnsan Hematopoetik Hücreler Hücre (CFC) Testi

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

colony-forming-cell-cfc-assay-for-human-hematopoietic-cells

Research

JoVE Journal

Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

35.5K Görüntüleme.  Washington University School of Medicine. Koloni oluşturan hücre (CFC) tayini hematopoetik atalarıdır yarı-katı ortamda koloniler oluşturur in vitro testtir. Koloni morfolojisi, hücre morfolojisi ve akış sitometri bir arada, farklı hematopoetik soy boyunca çoğalırlar ve ayırt atalarıdır yeteneklerini değerlendirmek için kullanılır.

Video: Colony Forming Cell CFC Assay for Human Hematopoietic Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus-spesifik T hücrelerinin üretilmesi Allojenik Hematopoetik Kök Hücre Nakli sonra Viral Enfeksiyonlar önleme / tedavi etmek için

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

İmmünoloji ve Enfeksiyon

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

Verimli HLA-Ig Tabanlı Yapay antijen sunan hücrelerin Ex vivo</emİnsan CTL> Genleşme

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

Human Papillomavirüs Roman T hücre epitopları Karakterizasyonu için interferon-γ Enzim bağlı immünospot Assay Kullanımı

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T Hücreleri İçin Kantitatif Yüksek throughput Tek hücreli Sitotoksisite Testi

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Saf Hematopoetik kök / progenitör hücrelerin DNA mutasyonları belirlenmesi

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Etkin Killer yapay antijen sunan hücreler (KaAPC)<em&gt; İn Vitro</em&gt; İnsan Antijen-spesifik T hücrelerinin tükenmesi

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Makrofaj koloni stimüle edici faktör farklılaştırılmış insan monosit türevli makrofajlar Kültür

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Akış Sitometresi tabanlı sitotoksisite tahlil insan NK hücre etkinliği değerlendirilmesi için

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...