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。

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		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 10mL Serological Pipet</td>
			<td>Corning</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 25mL Serological Pipet</td>
			<td>Corning</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL 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&#160; Clone&#160; 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)&#160;</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&#160; Clone&#160; M5E2</td>
			<td>BD Biosciences</td>
			<td>555398</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD209&#160; Clone&#160; 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 0.5M 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

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

科研

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...