Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Biz bu çalışmalarına katılmak için istekli olduklarını Miami WIHS çalışma katılımcılara teşekkür ederiz. Bu yazıda veriler Miami Kadın Kurumlararası HIV Çalışması (WIHS) ile toplanmıştır. Bu yayının içeriği sadece yazarların sorumluluğundadır ve Ulusal Sağlık Enstitüleri (NIH) resmi görüşlerini temsil etmemektedir. Bu çalışma İlerletme Allerji ve Enfeksiyon Hastalıkları Ulusal Enstitüsü ve Kadın Kurumlararası HIV enfeksiyonu Çalışması (WIHS) [hibe sayısı U01 AI103397], Ulusal Alerji ve Bulaşıcı Hastalıklar Enstitüsü, Sağlık [hibe sayısı P30AI073961] National Institutes, Ulusal Merkezi tarafından desteklenen Sağlık translasyonel Bilimleri ve Azınlık Sağlığı ve Sağlık Farklılıkların Ulusal Enstitüsü, Sağlık [hibe sayısı UL1TR000460 ve 1KL2TR000461] Ulusal Enstitüsü, Çocuk Sağlığı ve İnsan Gelişimi, Ulusal Enstitüsü Ulusal Enstitüsü [hibe sayısı K23HD074489].

Çalışma faaliyetleri Miami Kadın HIV Kurumlararası Çalışması (WIHS) ve AIDS Araştırma Miami Merkezi (SYAO) .Institutional İnceleme Kurulu (Üniversitesi Tıp Miami Miller Okulu) onayı öncesinde elde edilmiştir işbirliği ile Miami HIV Araştırma Birimi Üniversitesi&#39;nde gerçekleşti işe alma ve herhangi bir değerlendirme veya çalışma ile ilgili prosedürlere. 1. Endoservikal Fırça Örnek Toplama NOT: Katılımcılar eğitim MD ya da jinekolog ve cytobrush takarak spekulum muayenesinde altında yapıldı intraepitelyal lenfositler toplanması tarafından gerçekleştirilen bir vajinal muayene yapıldı. <ol><li> katılımcı özellikleri <ol><li> cinsel olarak aktif hamile değil ve kontraseptif ilaçlar veya rahim içi cihaz ile yaş, 18 ila 45 yaş arası kadınlar katılımcıları askere. </li><li> Vajinal muayenede önce, sahip katılımcıların yeşil bir 10 ml kan beraberlik geçmesiperiferik kan mononükleer hücre (hücre) izolasyonu için üst heparin vacutainers. endoservikal intraepitelyal analiz 9 için bir kontrol olarak hizmet vermektedir. </li></ol></li><li> Endoservikal örneklerinin toplanması <ol><li> yağlama olmadan vajina içine uygun büyüklükte bir steril spekulum yerleştirin. spekulum ve mukus temizlemek için steril gazlı bez sokulmasını kolaylaştırmak için ılık su kullanın. spekulum pozisyonu os ve ektoservikste tam görünüm için izin verir. </li><li> Ancak yakın sadece kıllar görünür olana kadar endoservikal kanala endoservikal fırça yerleştirin. 360 ° fırça saat yönünde çevirin. IMDM ortamı 5 ml ihtiva eden 15 mL&#39;lik bir tüp içerisine fırça aktarın. </li><li> Toplandıktan hemen sonra, buz üzerinde cytobrush içeren tüp yerleştirin. Toplanan intraepitelyal hücrelerinin yüksek canlılığını elde edilmesi için, 1 saat içinde ve laboratuar işleme cytobrush sağlar. </li></ol></li></ol><ol><li> 4 kısa (10 saniye) vuruş uygulayarak endoservikal cytobrush içeren vorteks tüp. </li><li> X 250 g 10 dakika süre ile, cytobrush içeren tüp santrifüjleyin. </li><li> Dikkatle tüpten cytobrush çıkarın (tüpün altındaki pelet rahatsız etmeyin) ve IMDM medya 1 ml ekleyin ve buz üzerinde tüp yerleştirin. </li><li> 50 ml tüp üstünde 100 mm hücre süzgecinden üzerinde cytobrush yerleştirin. </li><li> 20 ml IMDM ile cytobrush yıkayın. </li><li> 250 x g&#39;de 10 dakika boyunca santrifüj tüpü. </li><li> Süpernatantı Durusu ve pelet hücreler 1 ml IMDM tekrar süspansiyon ve 2.3 tarif zaten yeniden askıya pelet ile birleştirir. </li><li> Otomatik bir hücre sayacı 10 ile hücre sayımı veya tripan mavi boya, 10 uL hücre süspansiyonu 10 uL birleştirerek hemasitometre kullanarak hücreleri sayın. Yavaşça pipetlemeyin yukarı ve on kat aşağı hücreleri karıştırmak ve boya. Yük 10 uLİki hemositometre odaları birinin açıklığı içine karışımı. Beş ızgara alanlarında tüm hücreleri saymak. canlı ve cansız hücreler ayrı sayımı tutun. aşağıdaki formül kullanılarak hücre canlılığı belirlemek:% Canlı hücreler = cansız hücreler x 100 / toplam hücre sayısı hücreleri. Aşağıdaki hesaplamalar kullanarak ml başına müteakip hücre konsantrasyonu (ve hücrelerin toplam sayısı) belirler. ml başına toplam Hücreler = toplam hücre sayılan x (seyreltme faktörü / sayılan kare sayısı) 10.000 hücre / ml x. </li><li> % 10 DMSO ve% 90 FBS ihtiva eden dondurma ortamı kullanılarak izole endoservikal hücreleri dondurun. </li></ol> 3. Akım Sitometri <ol><li> PBS% 1 sığır serum albümini içeren 100 ul FACS tampon maddesi içinde 1-3 X 10 5 endoservikal hücreler (yeniden askıya </li><li> 1 uL / 10 Ekle 6 CANLI / DEAD fixable Sarı Ölü Hücre Leke kiti ve Tablo 1&#39;de açıklanan konsantrasyonlarda yüzey belirteçleri için antikorlar. </li><li> ışıktan korunan + 4 ° C sıcaklıkta 30 dakika boyunca inkübe hücreleri. </li><li> 350 xg 5 dakika için 1 ml FACS tampon ve hücreleri santrifüj tüpleri ekleme </li><li> 300 uL% 1 paraformaldehid hücre pelet yeniden askıya. </li><li> FACS tampon 100 ul boncuk bir damla ve örnek boyama için kullanılan antikorlara aynı konsantrasyonda eklenmesi ile boyanması için kullanılan boncuk ve antikorlar kullanılarak dengeleme kontrol hazırlayın. </li><li> sitometre 405 nm, 488 nm ve 635 nm lazer ile donatılmış bir akış lekeli boncuk ve örnekleri edinin. </li></ol> 4. endoservikal Gamma Delta T Hücre İzolasyonu <ol><li> Manyetik boncuk ayırma <ol><li> Yeniden askıya Malzemeler ve Reaktifler Tablo başvurulan TCR gama delta microbead kitine dahil tampon 40 uL, 2.6 anlatılan endoservikal hücre pelet (≤10 7 toplam hücreleri). </li><li> iki adımlı boyama protokolü için kit talimatları uygulayın: Birincisi, zekâanti-hapten mikroboncuklar-FITC H, anti-TCR y ö hapten antikoru ve ikinci </li><li> 4-8 ° C&#39;de 15 dakika inkübe edildikten sonra, 10 dakika boyunca 300 x g&#39;de tampon ve santrifüj 1 mL eklenerek hücrelerin yıkayın. Tersine tüp süpernatant süzün ve bir kağıt parçası yardımı ile kuru dokunun. </li><li> Resim tampon 500 uL hücre pelet yeniden askıya ve satıcının protokolüne göre, uygun manyetik ayırıcı yerleştirerek manyetik kolon hazırlanmıştır. </li><li> kolona hücre süspansiyonu uygulayın ve geçmesine etiketsiz hücreleri toplamak ve tampon uygun miktarda (500 uL) ile kolon 3x yıkayın. bir tampon üç kez, kolon rezervuar boş bir kez her zaman ekleyerek yıkama adımları uygulayın. toplam atık toplamak. Bu durum işaretlenmemiş, negatif hücre fraksiyonudur </li><li> Sütun sütun üzerine tampon 1 ml manyetik ayırıcı ve pipet oluşturmak ve hemen manyetik etiket ile fraksiyonu floş kaldırsıkıca kolonu ile birlikte piston uygulayarak hücreleri ed. NOT: manyetik olarak etiketlenmiş anti-hapten mikroboncuklar floresan boya FITC ile konjuge beri İzole gama delta T hücresi zaten floresan akış sitometri analizi için boyandı. </li></ol></li><li> hücre sıralama <ol><li> canlılığı kalıbı ve antikor paneli ile hücre sıralama, etiket endoservikal hücreler için bölüm 3&#39;de tarif edildiği gibi. </li><li> Canlı, CD45 +, CD3 + ve gama delta pozitif hücreler üzerinde elektronik kapıları kurmak ve hücre sıralama gerçekleştirin. </li></ol></li></ol>

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Yazarlar bir çıkar çatışması doğurabilecek ticari veya başka ilişki yoktur (örneğin, ilaç hisse senedi sahipliği, danışmanlık, danışma kurulu üyeliği, ilgili patentler ya da araştırma fonu).

kadın alt genital sistem mukoza açığına değerlendirilmesi, HIV edinimi ve bulaşma riskini ele klinik çalışmaların önemli bir bileşenidir. Tipik haliyle, HIV enfeksiyonuna FGT güvenlik genital salgıları (sitokinler, kemokinler ve antimikrobiyal peptitler) 12, 13 içinde çözünür immün modülatörler ölçerek değerlendirilir. Ancak, bu biyobelirteçler, vajinal iltihap sadece göstergesidir fizyolojik ve patolojik olaylardan etkilenir ve her ...

Biz intraepitelyal GD T hücrelerini değerlendirmek için endoservikal fırça örneklerini kullanarak metodolojisini geliştirdi. endoservikal kanal kolumnar epitel tek bir tabaka ile kaplı olduğu. İntraepitelyal lenfositler patojen mikropların 7, 8 karşılaşmak üzerine immün yanıtları hızla başlatabilir epitel tabakası içinde ikamet cephe lenfositler temsil etmektedir. endoservikal intraepitelyal bölmenin immünolojik olayların anlaşılması HIV de dahil olmak üzere hastalıklardan korunmak için etkili stratejiler tasarımı için önemlidir. Önerilen yöntem, bundan başka, HIV enfeksiyonu ya da HIV enfeksiyonu riski taşıyan kadınlarda intraepitelyal GD T hücrelerinin rolünün anlaşılması için çalışmalar yeni toplanmış insan endoservikal örneklerinde hem de dondurulmuş endoservikal hücreler için uygulanabilir. Bu protokolün ana amacı endoservikal intraepitelyal bölmede yeni immünolojik olayları keşfetmek ve değerlendirme, içinHIV enfeksiyonu olan kadınlarda mukozal açığına bir göstergesi olarak e GD T hücresi tepkileri. FGT bağışıklık etrafında bilgide önemli boşluklar başarıyla toplanması ve mukozal örnekleri işleme zorluk kısmen kaynaklanmaktadır. Ayrıca, karmaşık mukozal bağışıklık hücrelerinin heterojen ve birbirleri ile birbirine bağlılığı, bağışıklık sisteminin durumunu ve yeteneği yansıtan klinik olarak önemli ölçümler belirlenmesi için önemli zorluklar vardır. Biz daha olacak FRT dokunulmazlık yatan mekanizmaları belirlemek için kritik olabilir son derece mültipleksli, tek hücreli teknolojileri kullanılarak analiz edebilir yüksek derecede saflaştırılmış intraepitelyal GD T hücrelerini elde etmek için bir yöntem geliştirdi.

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

kadın üreme yolları (FRT) mukozal bağışıklık sistemi ilk savunma hattı olarak hizmet vermektedir. Genital mukozanın daha iyi bilgi HIV dahil olmak üzere farklı patojenlerin patojenitesini anlamak için elzemdir. Gama ö (GD) T hücreleri &#39;alışılmamış&#39; T hücrelerinin prototipi ve gamma ve delta zincirlerinden oluşan heterodimerik T hücre reseptörleri (TCR&#39;ler) kendi ekspresyonu ile tanımlanan T hücrelerinin görece küçük alt grubunu temsil eder. Bu alfa-beta TCR ile tanımlanır klasik ve daha iyi bilinen CD4 + yardımcı T hücreleri ve CD8 + sitotoksik T hücreleri ayıran. GD T hücreleri genellikle dokuya özel dağılımını gösteren ve epitelde zenginleştirilmiştir. GD T hücreleri iltihap, tümör gözetimi, enfeksiyon hastalıkları ve otoimmünite immün yanıtları düzenlemişlerdir. Burada, tekrarlanabilir izole ve insan endoservikal intraepitelyal GD T lenfositleri analiz etmek için bir yöntem mevcut. Biz haKadın Kurumlararası HIV enfeksiyonu Çalışması (WIHS) katılan kadınlardan kullanılan endoservikal cytobrush örnekleri ettik. vajinal mukoza hakaret var olduğu koşulları sırasında GD T hücreleri etkileşimleri hakkında bilgi vajinal microbicides.In ek geliştirilmesi dahil olmak üzere, mukozal açığı ele aldığı herhangi bir klinik çalışmada uygulanan olabilir, mukozal GD T hücre yanıtları hakkında bilgi vardır enfeksiyonlu hastalıkların tedavi edilmesi GD T hücre tabanlı immün terapinin uygulanması için potansiyeli.

Son zamanlarda, endoservikal intraepitelyal GD T hücrelerini analiz ve HIV bulaşmış kadınların 9, 11 yılında endoservikal gama delta T hücrelerinin kaybı bildiren ilk sendin. Burada, biz izole etmek ve endoservikal gama delta T hücrelerinin alt kümesini analiz protokol açıklar. Şekil 1 &#39;de gösterildiği gibi, GD T hücreleri hali hazırda insan endoservikal hücre örneklerinde tespit edilebil...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

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

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 ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.7K Görüntüleme.  Miller School of Medicine, University of Miami. 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.

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

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate İnsan Periferik Kan Genişleme γδ T Hücreler

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

İmmünoloji ve Enfeksiyon

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.

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 ...

Tıp

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

İzolasyon ve<em&gt; Ex Vivo</emVδ1 of&gt; Kültür<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T hücrelerinin bir Extrathymic αβT hücre Progenitör

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

İzolasyon ve Glioma-sızmakta periferik kan mononükleer hücrelerinin Akışı Sitometrik Analizi

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Görüntüleme Sitometrisi ile İnsan Monosit türetilmiş dendritik hücrelerle Karakterizasyonu: İki Monosit İzolasyon Protokoller Arasında Bir Karşılaştırma

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Önceden İşlem Görmüş Tümör Hücrelerinin Allojenik Ko-Kültür Ortamında İnsan T Hücresi Aktivitesinin Analizi

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Kanser Araştırmaları

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 ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

İki-fluorochrome tarafından yedi bağışıklık hücre alt kümeleri ayrımcılık Akış Sitometresi

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

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 ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

CD4 yalıtım+ T-hücreleri ve T foliküler Yardımcısı (cTfh) hücre alt kümeleri üzerinden periferik kan kullanarak 6-renk Akış Sitometresi Analizi, dolaşan

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

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