Name: collection, isolation, and flow cytometric analysis of human endocervical samples
Uploaded: 2014-07-06T15:00:00.000+00:00
Duration: 12 min 34 s
Description: Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Etik deyimi: Manitoba Üniversitesi&#39;nde ve Nairobi Kenyatta Ulusal Hastane / Üniversitesi&#39;nin hem araştırma etik kurulları bu çalışmayı onayladı ve yazılı bilgilendirilmiş onam tüm katılımcıların elde edilmiştir. 1.. Medya hazırlanması ve CMC Toplama Tüpleri <ol><li> Fosfat tamponlu tuzlu su (PBS) çözeltisi (137,93 mM NaCl, 2.67 mM KCI, 8.1 mM Na 2 HPO 4, 1,47 mM KH 2 PO 4) hazırlayın. Kısırlık için Otoklav. Bu birkaç ay boyunca 4 ° C &#39;de muhafaza edilebilir. </li><li> Ön kısım 5 ml PBS içine 50 ml şahin tüp (tahsil edilecek numune başına bir) ve 4 º C&#39;de mağaza örnek toplama zamanına kadar. </li><li> Depolama için lavaj toplamak için örnek başına 2-4 cryovials ya da 1.5 ml mikrosantrifüj tüpleri etiketleyin. </li><li> Hücre kültürü ortamı hazırlayın: RPMI 1640 +% 1 penisilin / streptomisin / amfoterisin B (nihai konsantrasyon 100 birim / ml, 100 ug / ml ve 250 ng / ml, respectivFCS yok / FBS&#39;li ely ekledi. </li></ol> Cytobrush Numune 2. Koleksiyonu Cytobrush örneklerinin toplanması eğitimli bir MD veya jinekolog tarafından yapılmalıdır non-invaziv bir işlemdir. <ol><li> Bir cytobrush ve bir tahta veya plastik kazıyıcı ikisi kullanılarak servikal mononükleer hücreleri (CMC) toplayın. (Şekil 1) sıyırıcıyı taktıktan ve servikal os etrafında dönen tarafından spekulum muayenesi altında katılımcıdan CMC&#39;yi toplayın. , Endoservikal os içine cytobrush yerleştirin 360 ° döndürmek ve derhal 5 ml PBS içeren 50 ml şahin tüp içine cytobrush ve kazıyıcı hem yerleştirin. </li><li> Işleme kadar 4 º C / buz örnekleri tutun. Toplama 2 saat içinde hücre canlılığı, süreç örnekleri korumak için. </li><li> Mümkünse, periferik kan mononükleer hücre CMC analizi için bir karşılaştırma / kontrol olarak hizmet etmek (hücre) izolasyon için yeşil üst heparin vacutainers eşleşen kan örnekleri toplamak. </li&gt; </ol> CMC 3. İzolasyonu Çift eldiven ile steril bir biyogüvenlik kabini içinde, bir biyogüvenlik düzeyi 2 laboratuarda örnek işleme gerçekleştirin. <ol><li> Toplama sırasında, CMC örnekleri kan ile kirlenebilir. Nihai numunede PBMC&#39;nin karıştırıcı dahil önlemek amacıyla çalışmadan görünür kan kirlenme örnekleri hariç. Nedeniyle CMC örneklerden izole edilen lenfositlerin düşük sayıda, küçük kan kontaminasyon kolayca CMC lenfosit bileşeni bunaltabilir. </li><li> Fırçalarından hücreleri ayırmak için 45 saniye boyunca cytobrush ve kazıyıcı hem de içeren Vortex şahin tüpler. Örnekler, bu işlem sırasında köpüklü hale gelebilir; Bu normaldir. </li><li> Kazıyıcı kapalı kalan malzemeyi kazımak ve bir çamaşır suyu çözeltisi içine kazıyıcı atmak için cytobrush kullanın. Fırça / kazıyıcı bağlı kalarak herhangi hücreleri toplamak için, başparmak ve işaret parmağı arasında kazıyıcı sıkmak taze bir eldiven kullanın.Hücreleri ve PBS aynı 50 ml tüp tahsil edilecek izin, fırçayı aşağı kaydırın. Bir çamaşır suyu çözeltisi içine cytobrush atmak ve eldiven atın. ÖNEMLİ: Her örnek için yeni bir eldiven kullanın. </li><li> Yeni bir 50 ml şahin tüp 100 mikron naylon hücre süzgeç yerleştirin. Bir transfer pipeti kullanarak, numune tüpten PBS-hücre süspansiyonu toplamak ve temiz bir tüp içine süzgeç içinden geçirilerek filtre edilmektedir. </li><li> Orijinal numune tüpüne RPMI 1640 içinde 5 ml. Transfer pipeti kullanarak, RPMI ile örnek tüpün tarafı yıkayın ve yeni bir toplama tüpüne filtreden aktarın. De RPMI ile naylon filtrenin alt yıkayın. </li><li> 514 x g de 10 dakika süre ile numuneler santrifüjleyin. Bu CMC pelet yerinden ayrılmasını önlemek amacıyla, bu aşama sırasında santrifüj fren bırakmak için çok önemlidir. </li><li> Yavaşça pelet rahatsız değil özen, tüpten çıkarın. Pelet boyutu son derece değişken olacaktır; öylesineDiğer durumlarda, topak zor görünür ve son derece şeffaf iken me örnekleri, topak nedeniyle epitel hücreleri çok sayıda varlığı oldukça büyüktür. </li><li> Falcon tüpün yumuşak çalkalama ile pelet yeniden süspanse edin. Örnek yıkamak için 5 ml PBS ekleyin. </li><li> Herhangi bir santrifüj freni ile 514 x g&#39;de 10 dakika boyunca tekrar santrifüj. </li><li> Dikkatlice süpernatant atın ve yukarıdaki gibi pelet yeniden askıya. Hücreleri (bir PBMC dondurulması protokolünü kullanarak) kriyokoruması, uyarma veya fenotiplen akış sitometri ya için hazırız. </li></ol> 4. CMC Yüzey Boyama ve Sitometrisi <ol><li> 100 ml bloke etme ul aşama 3.10 gelen pelet yeniden süspanse edin ve 96 oyuklu bir plaka ya da FACS tüpü içine ya aktarın. Bloklama çözeltisi (Fcy reseptörleri bloke etmek için) aşağıdaki tarifi: 1.8 ul fare IgG (nihai konsantrasyon 0.2 mg / ml), 5 ul FBS ve 93,2 ul FACS yıkama (PBS +% 2 FBS).Boyama topak boyutları kadar küçük ise, 96 oyuklu bir plaka içerisinde gerçekleştirilebilir, ama granül rutin olarak büyük ise FACS tüpler içinde gerçekleştirilebilir gerekebilir. Bu, buna göre antikor titre etmek için önemlidir. </li><li> 4 ° C&#39;de buz / 10 dakika boyunca numune bloke </li><li> 96 oyuklu plakalar içinde FACS yıkama (PBS +% 2 FBS), 100 ul, FACS ya da tüpler içinde 500 ul hücreleri yıkayın. Üzerinde düşük fren ile 4 ° C&#39;de 10 dakika boyunca 600 x g santrifüj (kapalı veya düşük fren ayarı kullanılamaz ise). </li><li> Süpernatantı ve ajitasyon ile hücre pelet yeniden askıya. Canlılığı Leke (Canlı Ölü düzeltilebilir canlılığı boya) ekleyin. Üreticinin talimatlarına uygun olarak canlılığı boya hazırlayın. Her laboratuvar / sitometresiyle kurulumunda kullanılmak üzere titre gerekebilir. 4 ° C&#39;de karanlıkta 30 dakika boyunca inkübe </li><li> (96 oyuklu plaka / FACS borular için) hücreleri PBS içinde 100 ul μl/500 yıkayın. Düşük / yok frenli 10 dakika boyunca 600 x g&#39;de santrifüj. Supernatan Kaldırt ve tekrar süspansiyon hücreleri. </li><li> 100 ul&#39;lik bir toplam hacimde, yüzey işaretleyici antikor kokteyli ile hücreleri inkübe edin. ÖNEMLİ: Optimize ve boyanma örnek önce panelde sitometri her akışını titre. 4 ° C&#39;de karanlıkta 30 dakika boyunca inkübe hücreleri </li><li> Adımı 4.5 tekrarlayın. bir FACS tüpü 96 oyuklu bir plaka, transfer hücrelerde boyama ve% 1 paraformaldehid (PFA) içeren FACS yıkama 750 ul&#39;lik nihai bir hacme kadar seyreltin (veya Cytofix 4, 1 &#39;seyreltilmiş). Bir akış Sitometre üzerinde veri toplama devam edin. </li></ol> 5.. Toplama ve Servikal Vajinal Lavajın Hazırlama (CVL) <ol><li> Örnekleme cytobrush önce, steril PBS içinde 2 ml endoserviksten yıkama ve posterior forniksten lavaj aspire için bir şırınga kullanın. </li><li> 15 ml şahin tüp içine aspire lavaj örnek toplamak ve işlenmesine kadar 4 º C / buz üzerinde tutmak. </li><li> Hücresel enkaz kaldırmak için 7 dakika boyunca 400 x g&#39;de santrifüj örnek. </li><li> Co-70 ° C&#39;de 1 ml ya da 500 ul alikotları ve deposuna süpernatan, kısım örnek llect Alikolar ELISA veya CVL proteinlerin boncuk dizisi analizi için çözülmüş, ancak birden donma / çözülme döngüleri önlemek olabilir. </li><li> Eğer arzu edilirse, üreticinin protokolü izlenerek RNA ekspresyonunu ölçmek için daha sonra RNA hücresel enkaz pelletini. </li></ol> 6.. Veri Toplama <ol><li> Antikor paneli maçlar tazminat tüpler bir dizi hazırlamak. Spektral örtüşme ayarlamak için tazminat tüpler çalıştırın. Bu sitometresi renkli bir akış örnekleri çalıştırın panelde kullanılan florokrom eşlenik antikorlar için yapılandırılır. </li><li> Lenfosit nüfusu tespit etmek için İleri Dağılım&#39;ı (FSC) ve yan dağılım (SSC) voltaj ayarlamak için ilk PBMC&#39;nin örnek edinin. Lineer ölçekte her bir eksen üzerinde 100 onları tutmaya çalışın. Bir PBMC denetimi Koşu önemli ölçüde daha kolay CMC lenfosit nüfusu bulmak için yapacaktır. </li><li> CMC numuneler için FSC eşik düşük FSC değerlerinde SSC eksenini smear nedeniyle PBMC&#39;nin numunelere göre artmış gerekebilir. </li><li> Almadan önce her tüp vorteks. Toplanan lenfosit kapısı olayların sayısını arttırmak için bütün boruyu edinin. Tıkanmasını önlemek için dikkatli makineyi izleyin. </li><li> Böyle FlowJo gibi bir akım sitometri analizi programına İhracat verileri. </li></ol> 7. Yolluk Stratejisi <ol><li> Yüksek İleri yan dağılım (FSC-H) seçin / İleri yan dağılım alanı (FSC-A) tekli nüfusu belirlemek için. </li><li> Edinimi kalitesi kontrol etmek için (örnek olarak FITC) Floresan işaretleyici karşı Saat&#39;i seçin. Akış hızı değişim verilerde eserler taşıyabilirler. Akış hızında farklılık gösteren herhangi bir alan kapsar. </li><li> SSC-A/FSC-A arsa üzerinde lenfosit popülasyonu tespit. Kimlik kolaylaştırmak için bir PBMC&#39;nin kontrolünü kullanın. CMC lenfosit nüfus PBMC kontrolü gibi net olmayabilir unutmayın. &lt;/ Li&gt; <li> SSC-A/viability üzerinde Kapısı canlı hücreler ölü hücreleri hariç boya. Ölü hücreler non-spesifik eserler tanıtacak olan antikorları dahil edebilirsiniz. </li><li> SSC-A/CD3 üzerinde kapı T hücre popülasyonu tespit etmek. Bu kapıdan, CD4 + ve CD8 + popülasyonlarının belirlenmesi. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+microscopy+and+ultrasound+imaging+of+human+cervical+structure.">Nonlinear optical microscopy and ultrasound imaging of human cervical structure.</a> Journal of biomedical optics. 18, (2013).</li><li>Oertelt-Prigione, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+the+menstrual+cycle.">Immunology and the menstrual cycle.</a> Autoimmunity reviews. 11, (2012).</li><li>Rahman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+serpin+A1+and+A3+levels+in+HIV+highly+exposed+sero-negative+women+are+affected+by+the+menstrual+cycle+and+hormonal+contraceptives+but+are+independent+of+epidemiological+confounders.">Mucosal serpin A1 and A3 levels in HIV highly exposed sero-negative women are affected by the menstrual cycle and hormonal contraceptives but are independent of epidemiological confounders.</a> American journal of reproductive immunology. 69, 64-72 (2013).</li></ol>

The authors have nothing to disclose.

Kadın genital sistem (FGT) de dokunulmazlık ile ilgili bilgide büyük boşluklar göz önüne alındığında, CMC fenotipik analizi serviks birden fazla lenfosit nüfus içine anlayışlar geniş bir dizi sağlayabilir. Proteomik analizi ve servikal lavajında ​​viral yük ölçümleri ile birleştiğinde, cinsel yolla bulaşan enfeksiyonlar (CYBE) ler ve diğer patojenler için bağışıklık çeşitli toplumlarda disseke edilebilir. Teknik hususlar - CMCs: izolasy...

Yeni HIV enfeksiyonlarının çoğunluğu dünya çapında kadınların 2011 yılında (UNAIDS 1) yeni enfeksiyonların% 47 temsil ile, heteroseksüel iletim yoluyla ortaya çıkar. Kadın genital (FGT), HIV ve diğer cinsel yolla bulaşan patojenler için ana giriş portallarından birini anlamak, enfeksiyonu önlemek için etkili stratejiler bulmak için yolda yüksek önem taşımaktadır. Genital mukoza bağışıklık karşılıkları da açık bir şekilde benzersiz ve periferik kan 2 ölçülen farklıdır. Ancak, FGT de bağışıklık dinamikleri mevcut bilgi en iyi sınırlıdır. Bugüne kadar, mukozal bağışıklık çevre çalışmaları büyük ölçüde enfeksiyonu takiben mukozal dokularda erken olaylar sonraki hastalığın ilerlemesi 3,4 üzerinde güçlü bir etkiye sahip olduğunu açık hale gelmiştir gut-ilişkili lenfoid dokularda (GALT), odaklanmıştır. Genital mukoza numune toplama büyük bir sorun teşkil etmektedir ve lak için en azından kısmen sorumludurFGT ve immünoloji anlayış k. FGT bu yerel örnekler toplamak ve analiz etmek için etkili yöntemler gerektiren olan farklı çevre bağlamında ev sahibi ve patojen arasındaki bağışıklık dinamiğin bulmaca çözme. FGT iki bölüme ayrılır: fallop tüpleri, endometrium ve endoserviksten ve ektoservikste içeren alt sistem ve vajina içeren üst üreme yolları (Kaushic tarafından gözden vd 5). Bu farklı sitelerde nispi katkısı HIV enfeksiyonuna ne hala belirsiz olduğunu, ancak her iki site HIV girişi 6 katkı verebilecek düşünülmektedir. T hücreleri, makrofajlar (Rodriguez-Garcia et al yorumlanan 2) yaklaşık 10% &#39;sini, üst ve alt üreme yolları olarak lökositlerin% 40-50 temsil eder. T hücreleri, vajina, rahim ve endometriyumda tespit edilebilir. Makrofajlar, daha güçlü olan localiher ikisi de dokularda tespit edilebilir, ancak, serviks daha endometriyum ve myometrial bağ dokusunda zed. Son olarak, plazmasitoid dendritik hücreler (PDC) ve Langerhans hücreleri de FGT dokularda tespit edilebilir. Fenotip ve bağışıklık nüfus ve HIV enfeksiyonuna karşı duyarlılık oranları hormonal döngülerine göre önemlisi değişebilir, hormonal kontraseptifler, bakteriyel vajinozis veya cinsel faaliyetler 5,7-9 kullanımı. Çeşitli yöntemler FGT bağışıklık nüfus ve çalışma ortamı için geliştirilmiştir. Servikal biyopsi, servikal cytobrushes ve servikovaginal lavajlar (CVL) 10-12 en sık literatürde genelinde kullanılmaktadır. PBS lavaj ile CVL toplama en kolay metodu olarak ve son derece düşük bir hücre verimle immün modülatör proteinlerin ancak sonuç çalışma sağlar ve bu nedenle FGT 13&#39;ün bağışıklık hücresi popülasyonlarının çalışmak için uygun değildir. CVL örnekleri, diğer taraftan, v olanELISA, sitokin boncuk dizi 14 ya da kütle spektrofotometresi 15,16 gibi yöntemler kullanılarak, çeşitli sitokinler, kemokinler ya da antimikrobiyal faktörlerin ekspresyonunu ölçerek FGT bağışıklık çevre değerlendirmek için yararlı ery. Immün hücre frekansları, fenotipler ve işlevleri karakterizasyonu servikal cytobrush veya servikal biyopsi örnekleme ile servikal mononükleer hücreleri (CMC) toplanması ile elde edilebilir. Servikal biyopsi örnekleme kanama rahatsızlık ve riskini artırır ve kadının 12 bağışıklık durumuna bağlı olarak prosedürü takip iyileşmek için 2 ila 11 gün sürer invaziv bir yöntemdir. Öte yandan, servikal cytobrushes toplandı, hücre alt verim rağmen FGT bağışık hücreleri toplamak için daha az invaziv ve daha uygun bir yöntemdir. Her iki yöntem de CD45 + lökositlerin aynı verimi ulaşabilir, ancak iki ardışık servikal cytobrushes ihtiva ettiği hücre aynı miktarda elde etmek için gerekli olan in bir biyopsi 13. Bununla birlikte, yine, 14 numune cytobrush akış sitometrisi ile daha da ex vivo feno hücre kabul edilebilir bir sayısı (yaklaşık 5000 CD45 + hücre / cytobrush) sağlamaktadır. Uyarılması ve hücre içi akış sitometrisi veya QPCR HIV spesifik immün tepkilerini 17 ya da hücre Th polarizasyonunu 18 tespit etmek cytobrush türevli CMC kullanılarak gerçekleştirilmiştir edilmiş olarak aynı zamanda, fonksiyonel karakterizasyonu, bu numuneler üzerinde ifa edilebilir. T hücreleri nüfusunun genişleme aynı zamanda CMC lerin 19 ile işlevsel çalışmalar kolaylaştırabilir. Bu biyopsiler ve cytobrushes FGT belirgin bölümlerini örnek olduğuna dikkat etmek önemlidir. Servikal cytobrushes endoserviksten ve muhtemelen transformasyon bölgesinin epitel türetilen hücrelerin toplanması, serviks örnek ise biyopsileri, ektoservikste 12,13 epiteli ve stroma üstün kısmından türetilmiştir. Cytobrush örnekler bu nedenle yeniden örnekbiyopsiler, bir skuamöz tabakalı epitelyum 5 ile kaplı bir bölge dahil ederken gion, kolumnar epitel tek bir tabaka oluşur. Bunun bir sonucu olarak, servikal biyopsi ve cytobrush tarafından toplanan lökosit popülasyonları doğası farklıdır. Cytobrushes CD14 + monositler / makrofajlar 13 daha yüksek bir oranda bir araya gelmesinden ise Biyopsiler, CD3 + T hücrelerinin daha yüksek bir oran toplamak. FGT immünolojisini incelenmesi uzun yıllar 20-22 bir ilgi oldu ve biz cytobrush türetilmiş CMC çalışma ile uzmanlık büyük bir birikimi var. Bizim çalışmalar, HIV ile enfekte enfekte ve HIV-seronegatif maruz Nairobi, Kenya (HESN) kadın seks işçileri çalışma çoğunlukla odaklanmak. HIV tercihli olarak FGT HIV tarafından hedeflenebilir aktive edilmiş hücre 23 ve daha düşük sayılar HIV satın karşı korunmasına katkıda bulunabilir, aktive edilmiş T hücrelerinde çoğaltır. Bu hipoteze göre, birkaç Studi doğrultusundaes henüz 24,25 enfekte kalır ve bu hareketsiz fenotip de FGT 14 gözlenen yüksek HIV maruz HESN seks işçileri arasında düşük bağışıklık aktivasyonunu tarif var. Burada, işleme için bir yöntem tarif ve ex vivo akış sitometrisi ile servikal cytobrushes türetilen CMC örneklerde T hücrelerinin aktivasyonunun değerlendirilmesi.

<table><tbody><tr><td>100 &amp;mu;m cell strainer for 50 ml Falcon tube</td><td>BD</td><td>352360</td><td>CMC processing</td></tr><tr><td>RPMI 1640</td><td>Hyclone</td><td>SH30027.01</td><td>CMC processing</td></tr><tr><td>Fetal bovine serum</td><td>Life technology</td><td>16000044</td><td>CMC processing</td></tr><tr><td>Fungizone</td><td>Life technology</td><td>15290-018</td><td>CMC processing</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>P4333-20ml</td><td>CMC processing</td></tr><tr><td>50 ml Falcon tube</td><td>Fisher</td><td>14-959-49A</td><td>CMC processing</td></tr><tr><td>Blood Bank disposable transfer pipette</td><td>Fisher</td><td>13-711-6M</td><td>CMC processing</td></tr><tr><td>Cytobrush plus</td><td>Cooper surgical</td><td>C0121</td><td>CMC sampling</td></tr><tr><td>Disposable cervical scraper</td><td>Quick medical</td><td>2183</td><td>CMC sampling</td></tr><tr><td>15 ml Falcon tube</td><td>Fisher</td><td>14-959-70c</td><td>CVL processsing</td></tr><tr><td>1.5 ml tube Eppendorf</td><td>Fisher</td><td>05-402-18</td><td>CVL storage</td></tr><tr><td>LIVE/DEAD Fixable Cell Stain Kit</td><td>Invitrogen</td><td>Various</td><td>Flow cytometry reagent</td></tr><tr><td>Fixation buffer (4% PFA)</td><td>BD</td><td>554655</td><td>Flow cytometry regeant</td></tr><tr><td>IgG mouse</td><td>Sigma</td><td>I8765</td><td>Flow cytometry regeant</td></tr></tbody></table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Multiparametreli akış sitometri önce karakterize dokularda hücre alt fenotipleri ve fonksiyonlarını incelemek için güçlü bir araçtır. CMC örneklerinin analizi, uygun yolluk stratejileri ile lenfosit ve monosit nüfus hem de bilgi verebilir. Temsili bir CMC yolluk bir strateji, bir eşleştirilmiş PBMC profili ile karşılaştırıldığında, Şekil 2 &#39;de gösterilmiştir. FSC-H grafiği FSC-A daha sonra filtrasyon, PBMC ile karşılaştırıldığında CMC...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Toplama, İzolasyon, ve İnsan endoservikal Akışı Sitometrik Analizi

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.1K Görüntüleme.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

Video: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Yalıtım Normal kolon ve kolorektal Karsinom - gelişmiş bir iletişim kuralı üzerinden insan endotel hücrelerinin

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Kanser Araştırmaları

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Kanserle İlgili Maddelerin Klinik Örneklerden Otomatik Olarak Ayrılması ve Toplanması

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Histerektomi Örneklerden İlköğretim İnsan endometrial stroma hücrelerinde Kurulması İçin İki Yöntem

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Tıp

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

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

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

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

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

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

İmmünoloji ve Enfeksiyon

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

Tek Hücreli Analizler için İnsan Fallop Tüpü Epitelinin Çıkarılması İçin Etkili Bir Yöntem

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo İnsan lenfoid doku ve erkek Genital mukoza insan immün yetmezlik virüsü 1 ve Histoculture ile enfeksiyonun

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

İnsan dişi üreme yolu dendritik hücreler yalıtım Phenotypical ve fonksiyonel çalışmaları için

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Akış Sitometrisi ile Analiz için Uterus Doğuştan Gelen Lenfoid Hücrelerin İzolasyonu

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

İnsan Sperm Fonksiyonel Kusurlarını Tespit Etmek için Biyobelirteçlerin Akış Sitometrik Analizi

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...