This work was supported by the Institute National du Cancer (INCa-DGOS-4664).

Primer lösemik hücreler ile 1. Nötrofil İzolasyon ve Co-kültür NOT: Prosedürler bilgilendirilmiş onam imzalanması tüm hastalarda Lyon Hastane Etik kurul onayı altında gerçekleştirilmiştir. <ol><li> Birincil lösemik hücreler ve nötrofillerin izolasyonu <ol><li> Kronik lenfositik lösemi (KLL) tanısı hastaların EDTA (kan mililitrede 1.8 mg EDTA) periferik kan tüpleri toplamak. </li><li> Bir steril 50 ml tüp kan her biri 15 ml ilave edilir ve 15 ml RPMI seyreltin (seyreltme 1: 1), daha sonra dikkatli bir şekilde yavaş yavaş fazlar karıştırılmadan tüpün dibine yoğunluk gradyan çözeltisi 15 ml ilave ediniz. yoğunluk gradyan çözeltisi hazırlanması için oda sıcaklığında olduğundan emin olun. </li><li> Oda sıcaklığında ve frensiz 35 dakika boyunca 300 xg&#39;de santrifüj. Şekil 1A, yukarıdan aşağıya doğru gösterildiği gibi kan dört farklı aşamadan ayırmak gerekir: trombosit ve plazma, mononükleer hücreleri (white halkası), yoğunluk gradyan çözümü, granülositler ve eritrositler. </li><li> Yeni 50 ml tüp plastik Pasteur pipeti ve transferi ile birincil lösemik hücreleri temsil beyaz halka toplayın. <ol><li> PBS ile tüp içinde oda sıcaklığında 10 dakika boyunca 300 g toplam ve santrifüj 50 ml&#39;ye kadar (kalsiyum ve magnezyum içerir) doldurun. </li><li> 5 PBS daha sonra oda sıcaklığında 10 dakika boyunca 300 g toplam ve santrifüj 50 ml&#39;ye kadar PBS ilave mL pelletini. </li><li> tam RPMI ortamı ile pelletini (RPMI 1640,% 10 fetal sığır serumu (FBS), 2 mM glutamin, 100 U ile takviye / ml penisilin ve 100 mg / ml streptomisin), hücre canlılığı sayacını kullanarak sayım için. </li></ol></li><li> Aspire Üst fazlar granülosit eritrosit faz ayrılıp 25 ml&#39;ye kadar PBS ekleyin toplam 50 ml% 0.9 NaClup içinde% 3 dekstran ekleyin. boruları 10 kez karıştırın ve karıştırma olmadan oda sıcaklığında 30 dakika boyunca muhafaza edin. </li><li> Üst RBC-fakir nötrofil katman toplayıntemiz ve steril bir 50 ml tüp içine yerleştirin ve oda sıcaklığında 10 dakika boyunca 300 g&#39;de santrifüj tüpleri d. PBS sonra toplam 50 ml kadar kırmızı hücre parçalama tampon ekleyin ml 5 ile her pelletini. </li><li> RT daha sonra oda sıcaklığında 10 dakika boyunca 500 g&#39;de santrifüj 15 dakika süreyle karanlıkta tüpler tutun. PBS ile pelet (toplam 50 ml&#39;ye kadar PBS ekleyin), daha sonra oda sıcaklığında 10 dakika boyunca 500 g&#39;de santrifüj yıkayın. </li><li> hücre canlılığı, sayılmadan için tam RPMI ortamı ile pelet yeniden süspanse edin. Akış sitometrisi kullanılarak izolasyon saflığını belirlemek için 5 ml&#39;lik plastik tüplere 50 ul PBS-FBS (% 4), her hücre türü için 3 x 10 5 tutun. </li></ol></li><li> İzole hücre popülasyonlarının Morfolojik Görüntüle&#39;ye analiz <ol><li> Bunu yapmak için, her biri 3 x 10 4 Nötrofiller ve 150 ul tam RPMI ortamında 3 x 10 4 mononükleer hücreleri yeniden süspanse edin. Bu t sağlanması, sitospin santrifüj cam slaytlar, filtre kartları ve örnek odaları birleştirinO santrifüj iyi dengelenmiş. </li><li> santrifüj slaytlar, filtre kartları ve örnek odalarında çıkarın oda sıcaklığında 10 dakika boyunca 750 x g&#39;de numune odasına ve hücre-santrifüje her bir hücre türü yer. slaytta hücrelere zarar vermemek için dikkatli sökün. Filtre kartları ve örnek odaları atın. </li><li> Giemsa boyama kiti kullanılarak hücreleri leke ve 1 saat kuruması için slaytlar tutun. 100X büyütme ile mikroskop hücreleri tarafından inceleyin. </li></ol></li><li> FACS İzole Hücrelerin Saflık test <ol><li> APC konjüge anti-insan CD19 antikoru (5 ul / 10 6 hücre) ile izole edilmiş, birincil lösemi hücreleri etiketlemek. Tablo 1 (5 ul / 10 6 hücre) listelenen anti-insan antikorlarının karışımı ile saflaştırılmış nötrofilleri etiketleyin. 4 O ° C&#39;de 30 dakika süreyle karanlıkta inkübe hücreleri </li><li> 500 ul PBS-FBS (% 4), oda sıcaklığında 5 dakika boyunca 300 g&#39;de santrifüj tüpleri hücreleri yıkayın. </li><li>200 ul PBS-FBS (% 4), her pelletini. </li><li> , SSC-A (488 / 10BP), FITC (BP 530/30), PerCP-Cy5.5 (695/40 BP FSC-A (488 nM): 488 nm lazer: Aşağıdaki optik yapılandırmayı kullanarak bir akış sitometresinde analiz ), PE (BP 575/26); 633 nm lazer: APC (BP 660/20), APC-Cy7 (BP 780/60). Renk karşılanmasını sağlamak. </li></ol></li><li> Otolog Nötrofiller ile İlköğretim lösemik hücrelerinin Coculture <ol><li> Tohum, birincil lösemi hücrelerinin tek başına ya da tam RPMI ortamı içinde oranı 1:10 otolog nötrofiller 2 x 10 5 hücre / ml olmuştur. Bunu yapmak için, seyreltilmesi hücre süspansiyonları, hücre sayıları ayarlayın ve 2 x 10 6 hücre / mL nötrofil birincil lösemi hücre 2 x 10 5 hücre / ml ekleyin. dikkatlice karıştırın. </li><li> hücrelere Bruton tirozin kinaz (Btk) inhibitörü (İbrutinib) 25 uM ekleyin. % 5 CO2 ile 37 ° C &#39;de 24 saat süreyle inkübe edilir. </li></ol></li><li> Hücre Hasat ve FACS analizi <ol><li> collECT, FACS analizi ve santrifüj oda sıcaklığında 5 dakika boyunca 300 g&#39;da tüpler 5 ml&#39;lik plastik tüplere hücreleri. daha sonra oda sıcaklığında 5 dakika boyunca 300 g&#39;de santrifüj 1 ml PBS-FBS (% 4) ile granül yıkayın. </li><li> ticari kit kullanılarak ve oda sıcaklığında 10 dakika boyunca karanlıkta inkübe hücreleri süspanse Annexin V ve PI peletler. (FSC-A (488 nM), SSC-A (488 / 10BP), FITC (BP 530/30), PI 610: 488 nm lazer: Şekil 2A yolluk stratejisi kullanarak bir akış sitometresinde ve aşağıdaki optik yapılandırmasına analiz / 20 BP). Renk karşılanmasını sağlamak. </li></ol></li></ol> Granulositik Pathway ve 3D Model RL Lenfoma B Hücreleri ile Bunların kokültürü boyunca HL60 Hücre 2. Farklılaşma <ol><li> Nötrofil benzeri Hücreleri içine insan promiyelositik (HL60) Hücre Farklılaşma <ol><li> / Ml daha sonra değişimi etkilemek için 1 uM retinoik asit (RA) ve% 1.25 DMSO ilave 3 x 10 5 hücre HL60 hücre sayısını ayarlar. Her 3 x 10 5 Tohum </skadar&gt; de 48 oyuklu plaka başına HL60 hücreleri daha sonra% 5 CO2 ile 37 ° C &#39;de 48 saat süre ile inkübe edilir. </li><li> Daha sonra, oda sıcaklığında 5 dakika boyunca 300 g hücreleri ve santrifüj toplar. hücre canlılığı, sayılmadan için tam RPMI ortamı ile tekrar süspansiyon hücreleri. </li><li> Sonra bu çözeltiye, 3 x 10 5 hücre / HL60 hücre sayısını ayarlamak ve 1 uM retinoik asit (RA) ve% 1.25 DMSO ilave edilerek kendi değişimini etkileme tam RPMI ortamı içinde hücre süspansiyonu seyreltin. Oyuk başına 48 oyuklu plaka içerisinde her biri 3 x 10 5 HL60 hücreleri, tohum ve% 5 CO2 ile 37 ° C sıcaklıkta bir 48 saat daha inkübe edilir. </li></ol></li><li> HL60 farklılaşma Analizi (HL60 fark) <ol><li> hücreleri toplamak ve oda sıcaklığında 5 dakika boyunca 300 g&#39;de santrifüj tüpleri. hücre canlılığı, sayılmadan için tam RPMI ortamı ile pelet yeniden süspanse edin. </li><li> flow sitometri ile hücre-yüzey belirteçleri ifade değişikliklerini analiz etmek. Her 3 x 10 5 koyun </sup&gt; ayrışmamış (hücre kültüründen) HL60 hücrelerinin ve FACS analizi ve santrifüj oda sıcaklığında 5 dakika boyunca 300 g&#39;da tüpler 5 ml&#39;lik plastik tüplere, 3 x 10 5 HL60 fark hücreleri. </li><li> 50 ul PBS-FBS (% 4) pelet yeniden süspanse edin. AF700 veya APC konjüge anti-insan CD38 (5 ul / 10 6 hücre) konjüge edilmiş bir anti-insan CD11b hücreleri etiketleyin. 4 O ° C&#39;de 30 dakika süreyle karanlıkta inkübe hücreleri </li><li> daha sonra oda sıcaklığında 5 dakika boyunca 300 g&#39;de santrifüj 500 ul PBS-FBS (% 4) ile yıkayınız. 200 ul PBS-FBS (% 4) pelet yeniden süspanse edin. </li><li> 488 nm lazer: Şekil 3A yolluk stratejisi ve aşağıdaki optik yapılandırma kullanarak akış sitometresinde analiz FSC-A (488 nM), SSC-A (488 / 10BP); 633 nm lazer: APC (660/20 BP), Alexa Fluor 700 (BP 730/45). </li><li> HL60 fark morfolojik değişiklikleri test etmek için, mikroskobik inceleme için cam slaytlar üzerine hücreleri hareketsiz. <ol><li> Bunu yapmak için, her biri 3 x 10 tekrar süspansiyon <su(hücre kültüründen) p&gt; 4 farklılaşmamış HL60 hücrelerinin 150 ul komple RPMI ortamı içinde 3 x 10 4 HL60 fark. santrifüj dengeli olmasını sağlamak, sitospin santrifüj cam slaytlar, filtre kartları ve örnek odaları birleştirin. </li><li> Oda sıcaklığında 10 dakika boyunca 1500 rpm&#39;de numune odasına ve hücre-santrifüje her bir hücre türü yer. santrifüj slaytlar, filtre kartları ve örnek odaları çıkarın. slaytta hücrelere zarar vermemek için dikkatli sökün. Filtre kartları ve örnek odaları atın. </li><li> Giemsa boyama kiti kullanılarak hücreleri leke ve 1 saat kuruması için slaytlar tutun. 100X büyütme ile mikroskop hücreleri tarafından inceleyin. </li></ol></li></ol></li><li> 3 boyutlu (3D) Kültür NOT: Bu deneyde kullanılan malzemeler soğuk ve deney buz üzerinde gerçekleşiyor emin olun. <ol><li> Tek başına veya HL60 fark ile karıştırılır ya her 5 x 10 4 RL hücreleri tekrar süspansiyon </em&gt; Oranı RL: HL60 fark 1:10 yaklaşık 2 ila 3 mm açıklığı genişletmek amacıyla steril makasla kesme, 1 ml bir pipet kullanarak 300 ul bazal membran matris ile karıştırılması. Bu adım sırasında kabarcıkları kaçının. </li><li> 24 oyuklu bir plaka içerisinde de / hücre süspansiyonu her biri 300 ul tohumu. Bu adım sırasında kabarcıkları kaçının. Daha sonra her bir RPMI ortamı doldurun ve% 5 CO2 ile 37 ° C &#39;de 7 gün süre ile inkübe ml 1 ekleyin% 5 CO2 ile 37 ° C&#39; de 30 dakika boyunca inkübasyona bırakılır. iki günde orta değiştirin. günde 5 10 nM vinkristin ekleyin. </li></ol></li><li> FACS analizi <ol><li> 7 günlük bir kültürden sonra ortam aspire ve iki kez 1 ml buz gibi soğuk PBS ile her bir yıkama. 3 ml / buz gibi soğuk PBS-EDTA (5 mM) da ekleyin. 200 ul pipet ucu alt kullanılarak kazıyarak Kuyunun dibinden jel ayırın. 30 dakika boyunca buz üzerinde hafifçe plakayı sallayın. </li><li> steril 15 ml tüp içine hücre süspansiyonu aktarın ve ic yavaşça tüpler sallayın30 dakika daha örn. homojen hücre süspansiyonu görünüm için kontrol edin. (Bu durumda değilse, o zaman daha uzun süre hücreleri sallamak ya da daha fazla PBS-EDTA ekleyin). </li><li> Oda sıcaklığında 10 dakika boyunca 300 x g&#39;de santrifüjleyin tüpler. PBS ile peletler daha sonra oda sıcaklığında 10 dakika boyunca 300 x g&#39;de santrifüj yıkayın. </li><li> PBS-FBS (% 4) ile yeniden süspanse edin ve PE-Cy7 ve APC konjüge anti-insan CD38 antikoru (5 ul / 10 6 hücre) bağlı anti-insan CD19 antikoru ile etiket. 4 O ° C&#39;de 30 dakika süreyle karanlıkta inkübe </li><li> Oda sıcaklığında 5 dakika boyunca 300 g sonra 500 ul PBS-FBS (% 4) santrifüj tüplerine ile yıkayınız. ticari kit kullanılarak Annexin V ve PI ile hücreleri tekrar süspansiyon. </li><li> Şekil 4A yolluk strateji kullanarak akış sitometresinde analiz ve aşağıdaki optik yapılandırma: 488 nm lazer: FSC-A (488 nM), SSC-A (488 / 10BP), FITC (530/30 BP), PI (610 / 20 BP), PE-Cy7 (780/60 BP); 633 nm lazer: APC (BP 660/20). Renk karşılanmasını sağlamak. </li></ol></li&gt; </ol>

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<li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophils+promote+the+malignant+glioma+phenotype+through+S100A4%5BTitle%5D)+AND+%22Clin+Cancer+Res+Off+J+Am+Assoc+Cancer+Res%22%5BJournal%5D)">Neutrophils promote the malignant glioma phenotype through S100A4.</a> Clin Cancer Res Off J Am Assoc Cancer Res. 20, 187-198 (2014).</li>
<li>Teramukai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pretreatment+neutrophil+count+as+an+independent+prognostic+factor+in+advanced+non-small-cell+lung+cancer%3A+an+analysis+of+Japan+Multinational+Trial+Organisation+LC00-03%5BTitle%5D)+AND+%22Eur.+J.+Cancer+Oxf.Engl+1990%22%5BJournal%5D)">Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00-03.</a> Eur. J. Cancer Oxf.Engl 1990. 45, 1950-1958 (2009).</li>
<li>Zhu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+IL-6-STAT3+axis+mediates+a+reciprocal+crosstalk+between+cancer-derived+mesenchymal+stem+cells+and+neutrophils+to+synergistically+prompt+gastric+cancer+progression%5BTitle%5D)+AND+%22Cell+Death+Dis%22%5BJournal%5D)">The IL-6-STAT3 axis mediates a reciprocal crosstalk between cancer-derived mesenchymal stem cells and neutrophils to synergistically prompt gastric cancer progression.</a> Cell Death Dis. 5, 1295(2014).</li>
</ol>

The authors have nothing to disclose.

yoğunluk gradyanlı santrifüj yaklaşımı kullanarak yüksek saflıkta insan kanından nötrofıller izolasyonu için ve aynı kademe tek çekirdekli hücreler içinde, burada etkili, basit, hızlı ve ucuz bir protokol açıklanan, aynı zamanda ayrılmakta ve geri kazanılmaktadır. izole edilmiş hücre popülasyonları ≥90% saftır. Çeşitli yöntemler insan kanından nötrofil izolasyonu için kullanılabilir. Bu süreksiz gradyanlar 11,12 kullanarak veya nötrofiller i...

Doğuştan gelen bağışıklık hücreleri, tümör mikro içindeki hücrelerin önemli bir kısmını oluşturmaktadır ve hastalar ve kanser 1 hayvan modellerinde tümör kanser ile ilişkilidir. Son zamanlarda, daha yaygın kronik immün yanıtları kemoterapiler 2 tümör ilerlemesi, metastaz ve direnç teşvik kritik bir rol oynayan takdir haline gelmiştir. Makrofajlar ile doğrudan kemoterapi 3,4 tümör hücresi yanıtı düzenleyen gösterilmiştir önemli doğuştan gelen bağışıklık hücrelerdir. Bununla birlikte, anti-kanser tedavisine, tümör yanıtı düzenleyen nötrofillerin, doğuştan gelen bağışıklık sisteminde anahtar oyuncular rolü bilinmemektedir. Bu protokollerin amacı KLL hastanın kan örneklerinden nötrofil ayırmak için hızlı ve güvenilir bir yöntem kullanmak ve anti-lenfoma ajanlara lenfoma hücrelerinin duyarlılığını düzenleyen rollerini araştırmak için granülositik yol boyunca HL60 hücreleri ayırt etmek vardır. 6 işgalci karşı ilk savunma hattı olarak kan 5 ve hareket doğuştan gelen bağışıklık sisteminin en bol hücresel bileşenidir. Nötrofiller, çeşitli patolojik şartlarda 7 değişken efektör fonksiyonlara ek olarak etkili bir doğuştan gelen bağışıklık yanıtları artan önemli bir rol oynamaktadır. Bu nedenle, yoğunluk gradyan ayırma yöntemi gibi diğer kan hücreleri, nötrofillerin izole etmek için, hızlı ve güvenilir bir yöntem olup, in vitro çalışmalar için gereklidir. Nötrofil izolasyonu için, bu yöntem kullanılarak, in vivo ve ex vivo nötrofil aracılığıyla oluşturulan imünolojik fonksiyonları üzerinde daha fazla araştırma kolaylaştıracaktır. Nötrofillerin saf popülasyonlarının elde etme yeteneği immünolojik hastalıkların 8 hastaların araştırılması için önemli bir ilk adımdır. Yoğunluk gradyan ayırma yöntemi hücrelerin yüksek verim elde edildiği bir yere bir tekniktir. methoD ara vermeden 35 dakika boyunca 300 g&#39;de santrifüje edilerek ve ardından seyreltilmiş insan kanı ihtiva eden bir tüpün alt yoğunluk gradyan çözeltisi ilave edilmesini kapsar. mononükleer hücrelerin halka arayüzü görünür ve nötrofiller eski aşağıda bulunur. Bu yöntem, çok daha 9 pahalı nötrofil izolasyon kiti gibi diğer mevcut yöntemlere göre önemli avantajlara sahip. Buna ek olarak, insan nötrofillerinden için spesifik bir yüzey işaretleyici yönelik antikorlar kullanılarak ticari kitler insan kanından nötrofilleri izole hücre aktivasyonunun veya farklılaşmasının riskini arttırır. Yoğunluk gradyan ayırma yöntemi kısa bir süre içinde, nötrofillerin izolasyonu sağlar. Aynı aşamada içinde, tek çekirdekli hücreler, ayrılmış ve geri kazanılır. Saf hücrelerin yüksek verimi fonksiyonel bütünlüğü elde etmek için elde edildiği bir temel teknik. Tümör microenv taklit etmek içinironment 3D deneyler gerçekleştirilmiştir. In vitro nötrofil kısa yarı ömürlü göz önüne alındığında, yeni insan nötrofil 3D deneyler kesin değildir. Bu nedenle, promiyelositik (HL60) hücreleri farklılaşma uyarıcılar dimetil sülfoksit (DMSO) ve retinoik asit (RA) kullanılarak, nötrofil benzeri hücrelere farklılaşmaları için indüklenir. Farklılaşmış HL60 hücreleri (HL60 fark) kullanarak, farklı donörlerden izolasyon nötrofillerin farklı tepkiler sahip önleyecektir. In vitro 3D kültür modelleri, in vitro 2D modelleri arasında ve in vivo modellerde bir ara evresini temsil eder. 2B kültüründe hücreler bu sentetik yüzey üzerinde denatüre edilir biriken proteinlere doğal olmayan hücre ekleri oluşturan plastik yüzeye yayılır. Bunun aksine, hücre ve hücre dışı matrisin sentezlenmesi için 3D kültürü da doğal hücre-hücre ek hücreler, bağlı oldukları doğal malzeme vardır.Bu nedenle, özellikle de, kanser hücreleri ve diğer hücre tipleri arasında 3D ko-kültür modelleri, tümör büyümesi, anjiyogenez ve metastazın katkıları gösteren için yararlı olmuştur. Bunun bir sonucu olarak, 3D kültürleri, hücre kültürü, in vivo 10 mevcut fizyolojik koşulları taklit etmek.

<table><tbody><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco Invitrogen</td><td>21875-034</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10270-106</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS contains calcium and magnesium)</td><td>Gibco Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>N-acetyl-L-alanyl-L-glutamine (L-Glutamine)</td><td>Life technologies</td><td>25030-024</td><td></td></tr><tr><td>Penicillin streptomycin (Pen Strep)</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Bruton&#039;s tyrosine kinase (Btk) inhibitor (Ibrutinib)&amp;nbsp;</td><td>CliniSciences</td><td>A3001</td><td></td></tr><tr><td>Vincristine&amp;nbsp;</td><td>EG labo</td><td></td><td></td></tr><tr><td>BD Matrigel basement membrane matrix&amp;nbsp;</td><td>BD Biosciences</td><td>354234</td><td>Put at 4 &lt;sup&gt;o&lt;/sup&gt;C overnight (O/N) before the day of the experiment</td></tr><tr><td>Red cell lysis buffer&amp;nbsp;</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Ficoll (Pancoll)</td><td>PAN Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Dextran&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>R2625</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Euromedex</td><td>S3014</td><td></td></tr><tr><td>Annexing V-FLOUS staining kit&amp;nbsp;</td><td>Roche</td><td>11&amp;nbsp;988&amp;nbsp;549&amp;nbsp;001</td><td></td></tr><tr><td>Kit RAL 555 Modified Giemsa staining kit</td><td>Cosmos Biomedical</td><td>CB361550-0000</td><td></td></tr><tr><td>LSRII flow cytometry</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Leica DMR-XA microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cellometer Auto T4 Cell Viability Counter</td><td>Nexcelom&amp;nbsp; Bioscience</td><td></td><td></td></tr></tbody></table>

neutrophil isolation and analysis to determine their role in lymphoma cell sensitivity to therapeutic agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

, Beyaz bir halka tek-çekirdekli hücreleri temsil eder, trombositler ve plazma. Burada tarif edilen yoğunluk gradyan ayırma yöntemi CLL hasta kanından izole primer lösemik hücreler ve uyarılmamış nötrofiller Şekil 1A, yukarıdan aşağıya doğru (yoğunluk gradyanlı santrifüj işlemi sonrasında elde edilen farklı kan katmanları temsil içerir yoğunluk gradyan çözeltisi, granülositler ve eritrositler). Şekil 1B ve 1C, sırasıyla n...

Watch this Scientific Journal Video about Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents at JoVE.com

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

neutrophil-isolation-analysis-to-determine-their-role-lymphoma-cell

Research

JoVE Journal

Immunology and Infection

15.2K Görüntüleme.  Cancer Research Center in Lyon. Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents. 

Video: Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Düşük yoğunluklu nötrofil tarafından oluşturulan nötrofil hücre dışı tuzakları Peritoneal lavaj sıvı aracılık tümör hücre büyümesini ve eki elde

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Kanser Araştırmaları

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Roman NFAT2 hedef genleri kronik lenfositik lösemi olarak tanımlamak için bir kromatin Immunoprecipitation tahlil

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

Birincil lösemi hücrelerinin metabolik profili değerlendirilmesi

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC tabanlı Testi İnsan Kronik Lenfositik Lösemi Hücrelerinde Ekstraselüler Nükleotid / Nükleozid Metabolizması Monitör

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Kimya

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Nükleer/Sitoplazmik Protein Ticaretini İzlemek Için Primer Kronik Lenfositik Lösemi Hücrelerinin Subsellüler Fraksiyonları

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

İnfiltratif Lenfositlerinin Analiz Olmayan enzimatik ayırmak Taze İnsan Dokusuna Basit ve Hızlı Protokolü

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

İmmünoloji ve Enfeksiyon

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Anti-tümör özelliklere sahip izole edilmesi ve nötrofil Karakterizasyonu

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Protein fonksiyonu için bir 2DE-Jel Spot: Kronik Lenfositik Lösemide HS1 öğrendim Ders

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Tıp

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Bir Optimize Protokol Lenfositlerindeki Glikoliz ve Mitokondriyal Solunum Analiz

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.