Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Menck, Kerstin; Behme, Daniel; Pantke, Mathias; Reiling, Norbert; Binder, Claudia R.; Pukrop, Tobias; Klemm, Florian

doi:10.3791/51554

Cited by 83 publications

(65 citation statements)

References 26 publications

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“…Peripheral blood derived monocytes (PBMCs) were isolated from human buffy coats by a Ficoll-Paque (1.077 g/cm 3 ) density gradient followed by a Percoll (1.131 g/cm 3 ) density gradient as previously described [39]. After both gradient separations, PBMCs were collected and washed with PBS containing 1mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation

et al. 2017

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Enhanced bioactive anti-oxidant formulations are critical for treatment of inflammatory diseases, such as atherosclerosis. A hallmark of early atherosclerosis is the uptake of oxidized low density lipoprotein (oxLDL) by macrophages, which results in foam cell and plaque formation in the arterial wall. The hypolipidemic, anti-inflammatory, and antioxidative properties of polyphenol compounds make them attractive targets for treatment of atherosclerosis. However, high concentrations of antioxidants can reverse their anti-atheroprotective properties and cause oxidative stress within the artery. Here, we designed a new class of nanoparticles with anti-oxidant polymer cores and shells comprised of scavenger receptor targeting amphiphilic macromolecules (AMs). Specifically, we designed ferulic acid-based poly(anhydride-ester) nanoparticles to counteract the uptake of high levels of oxLDL and regulate reactive oxygen species generation (ROS) in human monocyte derived macrophages (HMDMs). Compared to all compositions examined, nanoparticles with core ferulic acid-based polymers linked by diglycolic acid (PFAG) showed the greatest inhibition of oxLDL uptake. At high oxLDL concentrations, the ferulic acid diacids and polymer nanoparticles displayed similar oxLDL uptake. Treatment with the PFAG nanoparticles downregulated the expression of macrophage scavenger receptors, CD-36, MSR-1, and LOX-1 by about 20-50%, one of the causal factors for the decrease in oxLDL uptake. The PFAG nanoparticle lowered ROS production by HMDMs, which is important for maintaining macrophage growth and prevention of apoptosis. Based on these results, we propose that ferulic acid-based poly(anhydride ester) nanoparticles may offer an integrative strategy for the localized passivation of the early stages of the atheroinflammatory cascade in cardiovascular disease.

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Section: Methodsmentioning

confidence: 99%

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation

et al. 2017

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“…Utilizing specific surface markers in magnetic activated cell sorting (MACS) selection to purify monocytes from PBMC is feasible but this requires more facilities, indeed a remarkable proportion of monocytes might be missed due to restriction factors such as antigen-antibody cross reaction [6]. According to literature, monocyte purification based on their distinctive affinity of attachment can yield 95% purity [8,15].…”

Section: Discussionmentioning

confidence: 99%

“…To study the function of macrophages in vitro, the monocyte derived macrophage (MDM) is an acceptable and cost effective model [3][4][5]. Current protocols for MDM preparation require expensive process of monocyte purification by magnetic cell sorting (MCS) and needs colony stimulating factors such as granulocyte colony stimulating factor (GCSF) and granulocyte macrophage colony stimulating factor (GMCSF) for macrophage differentiation [6,7]. However, there are some more simple and economic methods for MDM culture and differentiation [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Culture and Differentiation of Monocyte Derived Macrophages Using Human Serum: An Optimized Method

Saghaeian-Jazi

Mohammadi

Sedighi

2016

Zahedan J Res Med Sci

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Background: Monocyte derived macrophages (MDMs) are appropriate in vitro models to study the function of macrophages in immune related diseases. Not only most of the methods in literature for efficient MDM culture and differentiation are expensive but also they require specific equipment. However, there are some reports indicating that monocyte enrichment is possible through attachment. Purification and differentiation of macrophages occurs in media containing human serum or platelet depleted plasma without extra supplementations. Different variables such as incubation time and serum concentration affect this simple MDM preparation method. Objectives: Here we represent an optimized simple method for MDM preparation from peripheral blood mononuclear cells (PBMC). Materials and Methods:To introduce an optimized method we accomplished the present descriptive study. After PBMC isolation and monocyte enrichment in complete RPMI 1640 growth media, macrophages were cultured and differentiated using human serum. Efficient phagocytosis was evaluated using heat-inactivated Escherichia coli followed by SYBR staining. Geimsa staining of macrophages was accomplished to visualize the typical morphology under light microscopy. Results: The derived macrophages have the typical morphology of differentiated macrophages and are able to phagocyte the heat inactivated SYBR stained E. coli. Conclusions: This optimized method is a simple and cost effective method to prepare MDM with typical morphology representations able to phagocytosis efficiently.

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“…Prepare the cord blood mononuclear cells (CBMCs) by density gradient centrifugation 14 per manufacturer’s instructions.…”

Section: Protocolmentioning

confidence: 99%

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Montoya

Ansel

2017

JoVE

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SHORT ABSTRACT The Neon® next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior. LONG ABSTRACT CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naive CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using the Neon® next generation electroporation device.

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Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Cited by 83 publications

References 26 publications

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation

Culture and Differentiation of Monocyte Derived Macrophages Using Human Serum: An Optimized Method

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

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