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>

<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.

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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

19.0K 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 Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Kısmen Pancreatectomized hastalar İnsan Islets izolasyonu

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Tıp

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

İnsan Pankreas için Toplama Protokolü

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

İnsan Fetal Pankreas Hücre Kümeleri İzolasyon, Kültür ve Görüntüleme

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Taze Cerrahi Örneklerden İzolasyon ve İnsan Ventriküler kardiyomiyositlerin Fonksiyonel Karakterizasyonu

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

İzolasyon ve profil MicroRNA içeren İnsan Bile dan ekzozomlar

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Farklı Homojenizasyon Yöntemleri ile Fare Beyin/Omurilikten Alınan Çoklu Hücre Tiplerinin Eşzamanlı Akış Sitometrik Karakterizasyonu

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

İnsan Kan Akışını Taklit Eden Akış Döngüsü Modelinde Kanla Temas Eden İmplantların Hemokomuuyum Testi

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Küçük Saç Örneklerinde 11 İlaca Dirençli TB İlaçların Ölçülmesi için Doğrulanmış LC-MS/MS Paneli

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

İnsan Viseral Yağ Dokusundan Canlı Adiposit ve Stromal Vasküler Fraksiyonun RNA Analizi ve Makrofaj Fenotiplemesine Uygun İzolasyonu

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...