We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies M&#252;hlbacher and Dr. Paul H&#246;rtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).

Etik deyimi: bilgilendirilmiş onam Yazılı Kan Transfüzyon &amp; İmmünolojik Bölümü, Innsbruck, Avusturya Merkez Enstitüsü tarafından tüm katılımcı kan vericilerden elde edildi. bilimsel amaçlarla anonim artık örneklerinin kullanılması Innsbruck Tıp Üniversitesi Etik Kurulu tarafından onaylandı. Periferal Kan Tek Çekirdekli Hücreleri 1. zenginleştirilmesi (PBMC) <ol><li> Santrifüj ile PBMC&#39;lerin konsantre. <ol><li> Alınan kan miktarına göre steril 50 ml santrifüj tüplerine kan paketi ve split kan açın. </li><li> Gerekirse, steril Dulbecco Fosfat Tamponlu Salin (D-PBS) ile her bir tüp için 50 ml göre ayarlayın. </li><li> Bir santrifüj tüpleri yerleştirilir ve fren oda sıcaklığında (RT) 15 dakika boyunca 400 x g&#39;de bunları dönerler. </li><li> boundar bırakarak, steril 10 ml serolojik pipet kullanarak plazma ve trombosit içeren üst katman kapalı Beraberliky tabakası rahatsız. Not: FCS yerine otolog plazma kültür ortamının ek kullanılırsa, plazma toplandı gerekir ve bu adımda ısı ile inaktive edilmiş. </li><li> iki adet 50 ml&#39;lik santrifüj tüplerine alınan mononükleer hücreler (sınır tabakaları) toplamak ve bir steril 10 ml serolojik pipet kullanılarak bir steril 50 ml tüp içine transfer edin. </li><li> Steril D-PBS kullanarak her bir tüp için 50 ml ses seviyesini ayarlayın. </li><li> Bir santrifüj tüpleri yerleştirin ve frensiz oda sıcaklığında 15 dakika boyunca 400 x g&#39;de onları dönerler. </li><li> bozulmamış sınır tabakası bırakarak, steril 10 ml serolojik pipet kullanarak plazma ve trombosit içeren üst katman kapalı çizin. </li><li> 50 ml santrifüj tüplerine alınan mononükleer hücreler (sınır tabakaları) toplamak ve bir steril 10 ml serolojik bir pipet kullanarak, iki steril 50 mi toplama tüpleri içine aktarın. </li><li> Steril D-PBS ile her tüp için 50 ml ses seviyesini ayarlayın. </li></ol></li><li> ayrılıkyoğunluk gradyan santrifüjü ile PBMC&#39;lerin (Şekil 1). <ol><li> Her tüpe yoğunluk gradyan ortamı dört adet 50 ml tüpler ve pipet 15 mi hazırlayın. </li><li> toplama tüpüne (adım 1.1.10) toplanan hücre / kan 25 ml alın ve dikkatle yoğunluk gradyan orta kaplaması. </li><li> Bir santrifüj tüpleri yerleştirilir ve fren RT&#39;de 30 dakika boyunca 700 x g&#39;de bunları dönerler. </li></ol></li><li> Toplama ve PBMC saflaştırılması. <ol><li> steril 10 ml serolojik pipet kullanarak plazma ve trombosit, rahatsız PBMC tabakası bırakarak içeren üst katman kapalı çizin. </li><li> Tüm tüplerden PBMC interfaz toplamak ve steril bir 25 ml serolojik pipet kullanarak, iki steril 50 ml tüpler hücreleri havuz. </li><li> Steril D-PBS ile her tüp için 50 ml ses seviyesini ayarlayın. Bir santrifüj tüpleri yerleştirilir ve fren ile, 4 ° C&#39;de 15 dakika boyunca 400 x g&#39;de bunları dönerler. </li><li> Süpernatantı aspire ve yeniden askıyaSteril, D-PBS içinde hücreleri. Her iki tüpe hücreleri havuz ve steril D-PBS ile 50 ml ses seviyesini ayarlamak. </li><li> santrifüj tüpleri yerleştirilir ve fren ile, 4 ° C&#39;de 10 dakika boyunca 400 x g&#39;de bunları dönerler. </li><li> Süpernatantı aspire ve steril D-PBS hücrelerin yeniden askıya. D-PBS 450 ul ihtiva eden 1.5 ml bir tüp steril D-PBS ile 50 ml ve pipet 50 ul hacim ayarlayın. </li><li> Vortex 1.5 ml tüp şiddetle ve Neubauer odasına veya diğer herhangi bir hücre sayım enstrüman hücreleri saymak. </li><li> santrifüje 50 ml tüpler yerleştirin ve fren ile, 4 ° C&#39;de 10 dakika boyunca 400 x g&#39;de bunları dönerler. </li></ol></li></ol> Anti-insan CD14 Manyetik Parçacıklar tarafından Monositler 2. izolasyonu <ol><li> Manyetik parçacıklar ile PBMC&#39;lerin etiketleme. <ol><li> Yalıtım tamponu kullanılarak 8 x 10 7 hücre / ml PBMC konsantrasyonu ayarlayın. </li><li> Vorteks anti-insan CD14 manyetik parçacıklar thoroughly ve her 1 x 10 7 PBMC&#39;ler için parçacıkların 50 ul ekle. </li><li> İyice hücre partikül süspansiyonu karıştırın ve 30 dakika boyunca oda sıcaklığında inkübe. </li></ol></li><li> Pozitif ve negatif fraksiyonların ayrılması. <ol><li> izolasyon tamponu kullanılarak 12 ml hacim kadar ayarlayın. </li><li> Her tüpe, hücre parçacık süspansiyonu altı steril yuvarlak tabanlı tüp ve pipet 2 ml hazırlayın. </li><li> Hemen mıknatıs tüpler yerleştirin. Oda sıcaklığında 10 dakika boyunca inkübe edin. </li><li> mıknatıs üzerinde tüpler tutun ve dikkatle süpernatant çizin. Süpernatan negatif kısmını içerir. </li><li> mıknatıstan tüpü çıkarın ve izolasyon tampon 2 ml ekleyin. </li><li> Hafifçe yukarı ve aşağı pipetleme hücreleri yeniden askıya. </li><li> geri mıknatıs üzerinde tüpler yerleştirin. Oda sıcaklığında 5 dakika süreyle inkübe edilir. </li><li> mıknatıs üzerinde tüpler tutun ve dikkatle süpernatant çizin. Süpernatan negatif kısmını içerir. </li><li>Mıknatıs tüpleri çıkarın ve her bir tüpe yalıtım tamponu 2 ilave edin. Hafifçe yukarı ve aşağı pipetleme hücreleri yeniden askıya. </li><li> steril bir 50 ml tüp pozitif kısmını aktarın ve steril D-PBS kullanarak 50 ml ses seviyesini ayarlamak. </li></ol></li></ol> IL-4 ve GM-CSF ile izole edilmiş monositlerin 3. uyarılması <ol><li> santrifüj tüpü yerleştirin ve fren ile 4 ° C&#39;de 10 dakika boyunca 400 xg&#39;de döndürün. </li><li> Aspire süpernatan ve D-PBS, 50 ml hücrelerin yeniden askıya. </li><li> D-PBS 450 ul içeren bir 1.5 ml tüp içine Pipet 50 ul. Vortex 1.5 ml tüp şiddetle ve Neubauer odasına ya da başka bir hücre sayım enstrüman hücreleri saymak. </li><li> santrifüj içine 50 ml tüp yerleştirin ve fren ile 4 ° C&#39;de 10 dakika boyunca 400 xg&#39;de döndürün. NOT: periferik kan lenfositleri (PBL) içeren negatif fraksiyon, ayrıca gerekiyorsa, yıkama gerçekleştirmek ve sayma t benzer adımlarPozitif fraksiyonun hortum (3.1-3.5 adımlar). </li><li> Önceden ısıtılmış bir kültür ortamı (RPMI 1640,% 10 ısı ile inaktive edilmiş FBS ve% 1 L-Glutamin çözeltisi ile desteklenen ortamda) ve pipet 3 ml / 6-çukurlu plakalara 1 x 10 6 / ml hücre yoğunluğu ayarlayın. NOT: ısı ile inaktive otolog plazma kullanıyorsanız, deneysel set-up göre,% 1-5 otolog plazma ile% 10 FCS değiştirin. </li><li> Kültür ortamı içine doğrudan sitokin pipetleme rekombinant insan IL-4 (250 U / ml) ve rekombinant insan GM-CSF (1000 U / ml) ile monositlerini uyarma. 37 ° C&#39;de inkübe edilir ve% 5 CO2. </li><li> Kültür ortamı içine doğrudan sitokin pipetleme 48 saat sonra IL-4 (250 U / ml) ve GM-CSF (1000 U / ml) ile hücrelerin yeniden teşvik eder. NOT: orta pH göstergesi ile gösterilen asitlenme herhangi bir işaret varsa, önceden ısıtılmış kültür ortamı ile orta yarısı değiştirin. </li><li> Hasat için, 5. günde dendritik hücreler olgunlaşmamışaşağı akım 6 oyuklu plaka üzerinden bir 50 ml santrifüj tüpüne kadar kültür ortamı pipetle sonra bir kaç kez aşağı kültürlenmiş hücreler pipetleme deneyler. </li><li> hücrelerin sayısı ve CD11b, CD11c karşı anti-insan antikorları izole monositler bir örnek leke ve birlikte bir hücre canlılığı boya ile DC-SIGN / CD209. Yüzey boyama sırasında antikorların özgül olmayan bağlanma önlemek için, reaktif ya da sığır serum albümini (BSA) tampon Fc reseptör bloke edici kullanılması önerilir. </li><li> Oluşturulan iDH&#39;lerin saflığını ve canlılığını değerlendirmek için akış sitometri gerçekleştirerek, üreticinin protokolüne göre, hücre canlılığı boya kullanılarak örnek analiz edin. </li></ol>

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</ol>

The authors have nothing to disclose.

Bu protokol, manyetik nanopartikül bazlı analiz kullanılarak anti-pıhtılaşmış kandan alınan insan monosit yalıtımla monosit türevli dendritik hücreler (MDDCs) üretimini tarif eder. Bu protokol, santrifüj adımları PBMC fraksiyonu bir zenginleşmeye yol açan hücre izolasyon prosedürü, üst baş tarafında ifa edilir. Hücreler santrifüj sırasında kaybolur, ancak, yoğunluk gradyanlı ortamının 200-300 ml gerektirecektir yoğunluk gradyan ortamında bütün bir kan paket içeriğini kaplamak için...

Dendritik hücreler (DC), bağışıklık sisteminin en önemli özel antijen sunan hücrelerdir. Olgunlaşmamış DC&#39;ler (iDH&#39;ler) deride veya mukozal dokularda bulunan ve işgalci patojenlere ile etkileşim ilk bağışıklık hücreleri arasında bu nedenle. Bu patojen tespiti, aşağıdaki T- ve B-hücresi tepkilerini aktifleştirme çünkü DH&#39;ler, doğal ve adaptif bağışıklık sisteminin 1 arasında bir köprü oluşturmaktadır. Bundan başka, çünkü bu tür IL-1, IL-6 ve IL-12 gibi sitokinler yüksek miktarda salgılanması pro-enflamatuar bağışıklık tepkilerinin katkıda bulunur. DH&#39;ler NK hücrelerini aktive ederek ve kemotaksi neden olduğu enfeksiyona sitesine başka bağışıklık hücrelerinin çeker. DH&#39;ler olgun olmayan dendritik hücreler (iDH&#39;ler) ve bunların yapısının ve fonksiyonunun göre olgun dendritik hücreler (MDC) 2 ayrılabilir. Birçok örüntü tanıma reseptörlerinin biri (örn, paralı-benzeri reseptörler, C tipi yabancı antijenlerin alımdan sonralektinleri veya bol hücre yüzeyi üzerinde ifade edilen) reseptörleri güzelleştirmek, iDH&#39;ler büyük değişiklikler geçmesi ve olgun başlar. Antijen sunumu için gerekli moleküllerin 3 yukarı regüle edilir ise, bu olgunlaştırma işlemi sırasında, antijen tutma reseptörleri, aşağı regüle edilir. Olgun DH&#39;ler ana histokompatibilite kompleksi I ve II (MHC I ve II) yukarı doğru regüle, T lenfositlerin antijen sunumu ve aktivasyonu için gerekli olan CD80 ve CD86 gibi eş-uyarıcı moleküller. Buna ek olarak, hücre yüzeyi üzerinde kemokin reseptör CCR7 ekspresyonu lenf düğümlerine periferik dokularda DC&#39;lerin geçişini sağlayan, indüklenir. Taşıma lenf düğümlerine 4-6 bir kemokin ligand 19 (CCL19 / MIP-3b) ve kemokin ligandı 21 (CCL21 / SLC) degrade boyunca DClerin &quot;döner&quot; ile kolaylaştırılır. Göç eden, MDC ve böylece ini, CD4 + ve CD8 + T hücreleri naiv işlenmiş antijeniistilacı patojen 7 karşı edinilmiş bağışıklık tepki tiating. Lenf düğümlerindeki T hücreleri ile bu etkileşimi de virüsü 8 yayılması ile ilişkilidir. Diğer in vitro çalışmalar DC&#39;ler verimli yakalamak ve güçlü bir enfeksiyon 9-12 Bu iletim sonuçlarına T hücrelerine ve HIV aktarmak olduğunu ortaya koydu. Bu deneyler lenf düğümlerine çevreden servisleri olarak in vivo HIV patlatır DC&#39;ler vurgulayın. Antijen sunumu sırasında DH&#39;ler efektör T yardımcı hücrelerinin farklılaşmasını şekil ve bu nedenle, mikrop karşı tüm bağışıklık tepkisinin sonucu bu çok etkileşim belirlenir önemli interlökinler salgılar. Yanı sıra tip 1 (Th1) ve tip 2 (Th2) efektör T hücreleri, CD4 + T yardımcı hücreleri (örneğin, tip 17 (Th17) ve tip 22 (Th22) T hücreleri) tarif edilmiştir ve diğer alt-gruplar ve bunların gidiş ve fonksiyon iyice incelenmiştir. DH&#39;ler, ayrıca katılandüzenleyici T hücreleri (Tregs) 13,14 nesil. Bu hücreler bağışıklık ve durdurmak ya da indüksiyon veya efektör T hücrelerinin çoğalmasını aşağı düzenleyen ve böylece bağışıklık ve hoşgörüyü geliştirmek için çok önemli olabilir. İnsan konvansiyonel DC&#39;ler (CDCS) bir miyeloid kökenli hücrelerin çeşitli alt kümelerini oluşturan (yani, Langerhans Hücreleri (LCs) ve dermal ve interstisyel DC&#39;ler) veya lenf kökenli (yani, plazmasitoid DC&#39;ler (PDC)). In vitro deneylerde veya DC aşılama stratejileri, monosit kökenli DH&#39;ler rutin dermal DC&#39;ler için bir model olarak kullanılmaktadır. Bu hücreler, geleneksel miyeloid dendritik hücrelerin fizyoloji, morfoloji ve fonksiyon benzerlikler göstermektedir. Bunlar, sağlıklı vericilerden 12,15-18 izole monositlere interlökin 4 (IL-4) ve granülosit-makrofaj koloni stimüle edici faktör (GM-CSF) eklenerek oluşturulur. Dendritik hücreler, doğrudan dermal ya da mukozal biopsiden izole edilmiş ya da olabilir b edilebilire CD34 + eski utero elde göbek kordonu kan örneklerinden izole edilen hematopoetik progenitör hücrelerden geliştirdi. Burada, monositler izole edilmiş ve yoğunluk dereceli santrifüj yoluyla periferal kan tek-çekirdekli hücresi (PBMC), zenginleştirme sonra anti-pıhtılaşmış insan kanından uyarılır gösterilmektedir. 5 gün için inkübe edildikten sonra, özel koşullar altında insan monositleri iDH&#39;ler ayrılırlar olmayan bir klinik deney prosedürleri için hazırdır.

<table><tbody><tr><td>APC Mouse Anti-Human CD19 Clone HIB19</td><td>BD Biosciences</td><td>555415</td><td></td></tr><tr><td>APC Mouse Anti-Human CD83 Clone HB15e</td><td>BD Biosciences</td><td>551073</td><td></td></tr><tr><td>BD Imag Anti-Human CD14 Magnetic Particles</td><td>BD Biosciences</td><td>557769</td><td></td></tr><tr><td>BD Imagnet</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BSA (Albumin Fraction V)</td><td>Carl Roth</td><td>EG-Nr 2923225</td><td></td></tr><tr><td>Costar 6 Well Clear TC-Treated Multiple Well Plates</td><td>Costar</td><td>3506</td><td></td></tr><tr><td>Density gradient media: Ficoll-Paque Premium</td><td>GE Healthcare Bio-Sciences</td><td>17-5442-03</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Falcon 10&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357551</td><td></td></tr><tr><td>Falcon 25&amp;nbsp;ml Serological Pipet</td><td>Corning</td><td>357525</td><td></td></tr><tr><td>Falcon 50&amp;nbsp;ml High Clarity PP Centrifuge Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Falcon Round-Bottom Tubes</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD3 Clone HIT3a</td><td>BD Biosciences</td><td>555339</td><td></td></tr><tr><td>Ghost Dye Violet 510 (Cell Viability Reagent)</td><td>Tonbo biosciences</td><td>13-0870</td><td></td></tr><tr><td>GM-CSF</td><td>MACS Miltenyi Biotec</td><td>130-093-862</td><td></td></tr><tr><td>Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America)</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>Hettich Rotanta 460R</td><td>Hettich</td><td>---</td><td></td></tr><tr><td>IL-4 CC</td><td>PromoKine</td><td>C-61401</td><td></td></tr><tr><td>Isolation buffer: BD IMag Buffer (10x)</td><td>BD Biosciences</td><td>552362</td><td></td></tr><tr><td>L-Glutamine solution</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.5 ml, SuperSpin</td><td>VWR</td><td>211-0015</td><td></td></tr><tr><td>PE Mouse Anti-Human CD14 Clone M5E2</td><td>BD Biosciences</td><td>555398</td><td></td></tr><tr><td>PE Mouse Anti-Human CD209 Clone DCN46</td><td>BD Biosciences</td><td>551265</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Sigma-Aldrich</td><td>R0883</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr></tbody></table>

generation of human monocyte-derived dendritic cells from whole blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Bir sükroz yastık kullanılarak anti-pıhtılaşmış kanın santrifüje edilmesinden sonra, periferal kan tek-çekirdekli hücreleri (PBMC), yoğunluk gradyan ortamı (Şekil 1) üstüne bir arayüz içinde zenginleştirilmiştir. PBMC&#39;ler çekilir sonra FACS analizi soyu belirteçler kullanılarak PBMC (örneğin, CD3 T lenfositlerin, CD14 monositler için ve CD19 B lenfositleri için) olan farklı hücre popülasyonları için gerçekleştirilir. <stron...

Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

generation-of-human-monocyte-derived-dendritic-cells-from-whole-blood

Research

JoVE Journal

Immunology and Infection

20.2K Görüntüleme.  Medical University of Innsbruck. Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Video: Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritik hücreler kemik iliği öncüleri Kültür

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biyoloji

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Kemik İliği Üretimi 2-foton Görüntüleme Kullanım murin Dendritik Hücreler Türetilmiş

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

İmmünoloji ve Enfeksiyon

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Metabolik fenotipleri farklı olarak sahip Olgunlaşmamış, Olgun ve tolerojenik Dendritik Hücrelerinin Üretimi

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Çok sayıda miyeloid ataları ve dendritik hücre öncüleri fare kemik iliği strateji sıralama bir roman hücre kullanarak gelen nesil

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Sığır Monosit Kaynaklı Dendritik Hücreler Kullanılarak Aşı İmmünojenitesinin Belirlenmesi

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

Sialidaz Tedavisi Kullanılarak İzole PBMC'lerden De-Sialillenmiş Monosit Türetilmiş DC'ler

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Yazar Spotlight: Hücre Yüzeyinin Sialidaz Bazlı Enzimatik İşlemi Kullanılarak Dendritik Hücrelerin Olgunlaşması 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Farklı Sialillenmiş Fenotiplere Sahip Monosit Kaynaklı Dendritik Hücrelerin Üretimi

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

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