Name: generation of human monocyte-derived dendritic cells from whole blood
Uploaded: 2016-12-24T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Generation of Human Monocyte-derived Dendritic Cells from Whole Blood at JoVE.com

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).

伦理学声明:书面知情同意从所有参与献血中央研究所输血与免疫学系，奥地利因斯布鲁克获得。使用匿名剩余标本用于科学目的的批准因斯布鲁克医科大学伦理委员会。 1.富集外周血单个核细胞（PBMC中） <ol><li> 通过离心外周血单个核细胞的浓度。 <ol><li>根据血液中收到的金额开血包和分裂的血液在无菌的50ml离心管中。 </li><li>如果需要的话，调节音量至50ml对于每个管，用无菌的Dulbecco磷酸盐缓冲盐水（D-PBS）中。 </li><li>管放入一个离心机并在400 xg离心无制动旋转它们在室温（RT）下15分钟。 </li><li>抽出用10ml无菌血清吸管含有血浆和血小板的上层，留下boundary层的干扰。 注意:如果自体血浆代替FCS的用于补充培养基中，等离子体必须收集并在此步骤中热灭活。 </li><li>从两个50毫升离心管中收集的单核细胞（边界层），并使用10ml无菌血清吸管将其转移到一个无菌的50ml管中。 </li><li>调节音量至50ml，使用无菌的D-PBS每个管。 </li><li>管放入一个离心机，并在室温在400 xg离心旋转他们15分钟无制动。 </li><li>抽出含有用10ml无菌血清吸管，而使边界层不受干扰的血浆和血小板的上层。 </li><li>从50ml离心管中收集的单核细胞（边界层），并转移到用10ml无菌血清吸管疏导50毫升收集管。 </li><li>调节音量至50ml对于每个管，用无菌的D-PBS中。 </li></ol></li><li> 分割通过密度梯度离心的PBMC（图1）。 <ol><li>准备密度梯度介质的四五十毫升管和吸管15毫升到每个试管中。 </li><li>需要25毫升汇集细胞/血液从收集管（步骤1.1.10），并小心地覆盖密度梯度介质。 </li><li>管放入一个离心机，并在室温以700 xg离心旋转他们30分钟无制动。 </li></ol></li><li> 收集和外周血单个核细胞的纯化。 <ol><li>抽出含有用10ml无菌血清吸管的血浆和血小板，留下PBMC层不受干扰上层。 </li><li>收集所有的管子相间PBMC并用无菌25毫升血清吸管集中细胞有两种无菌50ml试管。 </li><li>调节音量至50ml对于每个管，用无菌的D-PBS中。管放入一个离心机并在400 xg离心在4℃下与制动旋转他们15分钟。 </li><li>吸出上清液并重新悬浮细胞在无菌的D-PBS。汇集来自两个管中的细胞，并调节音量至50ml，用无菌的D-PBS中。 </li><li>管放入离心机并在400 xg离心在4℃下与制动旋转他们10分钟。 </li><li>吸出上清液并重新悬浮于无菌的D-PBS中的细胞。调节音量至50ml，用无菌的D-PBS和吸管50微升含有450微升的D-PBS的1.5ml试管。 </li><li>涡1.5 ml管大力和计数在纽鲍尔室或任何其他细胞计数仪细胞。 </li><li>在50ml试管放入离心机并在400 xg离心在4℃下与制动旋转他们10分钟。 </li></ol></li></ol> 2.分离单核细胞的通过抗人CD14的磁颗粒<ol><li> 与磁性粒子外周血单个核细胞标记。 <ol><li>调整PBMC浓度使用隔离缓冲器8×10 7细胞/ ml。 </li><li>涡旋抗人CD14磁性粒子thoroughly和加入50微升的颗粒，每1×10 7外周血单个核细胞。 </li><li>彻底混合细胞颗粒悬浮液，并在室温下孵育它为30分钟。 </li></ol></li><li> 正和负级分的分离。 <ol><li>调节音量使用隔离缓冲区12毫升</li><li>制备细胞颗粒悬浮六无菌圆底管和移液管2毫升到每个管中。 </li><li>立即放置在磁体的管中。孵育它们在室温10分钟。 </li><li>保持磁铁管并仔细清除表面。上清液中含有的阴性部分。 </li><li>从磁铁取出管加2ml分离缓冲。 </li><li>通过上下吹打轻轻重新悬浮细胞。 </li><li>放置管背面上的磁铁。在室温下孵育5分钟。 </li><li>保持磁铁管并仔细清除表面。上清液中含有的阴性部分。 </li><li>从磁体取出管和加2ml隔离缓冲液到每个管中。通过上下吹打轻轻重新悬浮细胞。 </li><li>转移阳性分数到无菌的50ml管中，并调整体积至使用无菌的D-PBS为50ml。 </li></ol></li></ol> 3.隔离单核细胞的刺激与IL-4和GM-CSF <ol><li>放置管进入离心机并在400 xg离心在4℃下与制动旋转它10分钟。 </li><li>吸出上清液并重新悬浮细胞在50毫升的D-PBS中。 </li><li>吸管50微升到含有450微升的D-PBS的1.5ml试管。涡1.5 ml管大力和计数在纽鲍尔室或其他细胞计数仪细胞。 </li><li>放置50毫升管进入离心机并在400 xg离心在4℃下与制动旋转它10分钟。 注:如果阴性部分，含有外周血淋巴细胞（PBL），也是必需的，进行洗涤及数量的步骤类似于t阳性部分的软管（步骤3.1-3.5）。 </li><li>调节细胞浓度为1×10 6个 / ml的带预热的培养基（补充有10％热灭活FBS和1％L-谷氨酰胺溶液的RPMI 1640培养基）和移液器为3ml /孔到6孔平板上。 注意:如果使用热灭活的自体血浆，替换1-5％自体血浆的10％FCS，根据实验设置。 </li><li>通过直接吹打细胞因子至培养基用重组人IL-4（250 U / ml）和重组人GM-CSF（1000单位/毫升）刺激单核细胞。在37℃和5％的CO 2。 </li><li>通过直接吹打细胞因子至培养基中重新刺激与IL-4（250 U / ml）和GM-CSF（1000U / ml）的细胞后48小时。 注意:如果存在由在介质中的pH指示剂所示酸化的任何迹象，预热培养基替换一半培养基。 </li><li>收获未成熟树突状细胞在第5天进行下游吹打培养细胞进行的6孔板和到50毫升离心管中向上吸取培养基后和向下几次实验。 </li><li>计数细胞并染色用抗CD11b的，CD11c的抗人抗体中分离单核细胞的样品，和DC-SIGN / CD209，具有细胞活力染料一起。为了避免表面染色过程中的非特异性的抗体的结合，强烈建议封闭试剂或牛血清白蛋白（BSA）的缓冲区的使用Fc受体的。 </li><li>分析使用细胞生存力染料样品，根据生产商的方案，通过进行流式细胞术来评估所产生的iDC的纯度和生存能力。 </li></ol>

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The authors have nothing to disclose.

这个协议描述通过使用磁性纳米微粒测定从抗凝固的血人单核细胞的分离单核细胞来源的树突细胞（MDDCs）的产生。在这个协议中，在离心步骤上游的细胞分离方法，这导致PBMC部分的富集进行。虽然细胞离心过程中丢失，叠加密度梯度介质上的全血包的内容将需要200-300密度梯度培养基中，因此不能被认为是成本有效的。此外，的PBMC通过在清洁的细胞群，在分离过程产生积极影响的抗人CD14磁性颗?...

树突细胞（DC）是免疫系统中最重要的专门的抗原呈递细胞。未成熟DC（iDC中）驻留在皮肤或粘膜组织，因此是所述第一免疫细胞与入侵的病原体相互作用之间。的DC代表先天和适应性免疫系统1之间的桥梁，因为它们可激活下列病原体检测T细胞和B细胞反应。此外，它们有助于由于大量的细胞因子，如IL-1β，IL-6，和IL-12的分泌的促炎性免疫应答。 DCS还可以激活NK细胞，并吸引其他免疫细胞对感染的趋化网站。 的DCs可分为未成熟树突状细胞（iDC中），并根据它们的形态和功能的成熟树突状细胞（的mDC）2。由（许多模式识别受体之一例如 toll样受体，C型识别外来抗原的后凝集素，或补体受体）在细胞表面上大量表达，将iDC发生重大的变化，并开始成熟。在此成熟过程中，受体抗原捕获被下调，而对于抗原呈递必需分子被上调3。成熟DCs上调主要组织相容性复合物I和II（MHC I和II），共刺激分子象CD80和CD86，这对于T淋巴细胞的抗原呈递和活化所必需的。此外，在细胞表面上的趋化因子受体CCR7的表达被诱导，这使得DC的从外周组织至淋巴结的迁移。迁移由沿趋化因子配体19（CCL19 / MIP-3b）的与趋化因子配体21（CCL21 / SLC）梯度到淋巴结4-6 DC的"滚动"促进。 移植完成之后，MDC在目前的处理抗原天真的CD4 +和CD8 + T细胞，从而INItiating对抗入侵病原体7适应性免疫应答。这与在淋巴结的T细胞相互作用也与病毒8的传播有关。 其他体外研究表明，树突有效地捕捉和HIV一场轰轰烈烈的感染9-12转移到T细胞和这个传输的效果。这些实验强调， 在体内的HIV攻击的DCs作为从外围到淋巴结梭。期间抗原呈递树突分泌塑造效应T辅助细胞的分化，并且因此，对微生物的整个免疫反应的结果，在这个非常相互作用判定键白细胞介素。除了1型（Th1细胞）和2型（Th2细胞）的效应T细胞，已经描述的 CD4 + T辅助细胞（ 例如，类型17（Th17细胞）和类型22（TH22）T细胞）的其他子集，以及他们的诱导和功能已经被彻底调查。区议会进一步参与产生调节性T细胞（Treg细胞）13,14。这些细胞具有免疫抑制作用，可以停止或下调诱导或效应T细胞的增殖，因而制定免疫力和宽容的关键。 人类传统的DC器（CDC）包括细胞几种亚型与骨髓来源（即朗格汉斯细胞（LCS）和真皮和间质的DC）或淋巴来源（即浆的DC（pDC细胞））。对于体外实验或DC疫苗接种策略，单核细胞衍生的DCs被常规用作用于皮肤的DCs的模型。这些细胞显示在生理，形态和功能与常规骨髓树突状细胞的相似性。它们通过加入白介素-4（IL-4）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）与来自健康供体12,15-18分离的单核细胞产生的。树突细胞也可以直接从皮肤或粘膜活检分离，或可以甚至bE从CD34 +从获得的子宫外脐带血样本中分离出的造血祖细胞的发展。在这里，我们证明单核细胞是如何分离和密度梯度离心的外周血单核细胞（PBMC）富集之后，从抗凝固的人血的刺激。培养5天后，在特定条件下的人单核是分化成的iDC，并准备用于在非临床环境实验程序。

<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.

的使用蔗糖缓冲抗凝血离心分离后，外周血单核细胞（PBMC）富集在密度梯度介质（ 图1）的顶部的相间。在PBMC中被抽出后，被执行以使用谱系标志物的PBMC中（ 例如，CD3对T淋巴细胞，CD14为单核细胞和CD19为B淋巴细胞）中表征不同细胞群体的FACS分析。 图2A示出了收集和密度梯度离心后染色的PBMCs的代表性FACS分析的结果。出选通人口（ ?...

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.

从全血人单核细胞来源的树突状细胞的生成

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

20.1K 次观看.  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.

视频: 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.

文化髓系树突状细胞从骨髓前体

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

科研

生物学

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.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

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).

隔离和小鼠骨髓的静脉注射单核细胞衍生

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

免疫与感染

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.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

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.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

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.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

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.

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

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

使用唾液酸酶处理从分离的 PBMC 中去除唾液酸化单核细胞衍生的 DC

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.

作者聚焦:使用基于唾液酶的细胞表面酶处理树突状细胞成熟 

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.

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

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

从全血人单核细胞来源的树突状细胞的生成

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文化髓系树突状细胞从骨髓前体

代骨髓衍生的双光子成像使用的小鼠树突状细胞

隔离和小鼠骨髓的静脉注射单核细胞衍生

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成