Isolation of Human Monocyte Populations

Wahl, Larry M.; Wahl, Sharon M.; Smythies, Lesley E.; Smith, Phillip D.

doi:10.1002/0471142735.im0706as70

Cited by 54 publications

(45 citation statements)

References 7 publications

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“…The monocyte fraction was purified by counterflow centrifugal elutriation, as previously described (21,22), and contained Ͼ90% monocytes, as determined by morphology and flow cytometry. Monocytes were cultured in serum-free DMEM (Cambrex) supplemented with 2 mM L-glutamine (Mediatech) and 10 g/ml gentamicin (Cambrex).…”

Section: Purification and Culture Of Human Monocytesmentioning

confidence: 99%

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin

Zhang¹,

Zhou²,

Bugge

et al. 2007

The Journal of Immunology

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Chronic inflammatory diseases are associated with connective tissue turnover that involves a series of proteases, which include the plasminogen activation system and the family of matrix metalloproteinases (MMPs). Urokinase-type plasminogen activator (uPA) and plasmin, in addition to their role in fibrinolysis and activation of pro-MMPs, have been shown to transduce intracellular signals through specific receptors. The potential for uPA and plasmin to also contribute to connective tissue turnover by directly regulating MMP production was examined in human monocytes. Both catalytically active high m.w. uPA, which binds to the uPAR, and low m.w. uPA, which does not, significantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the N-terminal fragment of uPA, which binds to uPAR, but lacks the catalytic site, failed to induce MMP-1 production, indicating that uPA-stimulated MMP-1 synthesis was plasmin dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2, as demonstrated by inhibition of MMP-1 production with Abs against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of ERK1/2 and p38 MAPK, cyclooxygenase-2, and PGE2, leading to increased MMP-1 production. Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin induction of MMP-1, suggesting that inactive plasmin may be useful in suppressing inflammation.

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Section: Purification and Culture Of Human Monocytesmentioning

confidence: 99%

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin

Zhang¹,

Zhou²,

Bugge

et al. 2007

The Journal of Immunology

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“…Monocytes distinguishable ability for attachment makes it possible to purify them from PBMC simply [14,15]. Different variables could affect the efficiency of purification [5,8,9,11,14,16].…”

Section: Resultsmentioning

confidence: 99%

“…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]. Some researchers have reported gelatin coating or fibronectin coating [19] and plasma treatment the surface of culture plates can facilitate the attachment [12] while we found no difference between monocyte attachment enrichment in gelatin or plasma treated and non-treated tissue culture plates.…”

Section: Discussionmentioning

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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“…One has to be aware of other methods that allow isolation of primary human monocytes: adherence to plastic, positive bead isolation, or counter flow centrifugation elutriation 17 . The monocyte enrichment cocktail used here contains antibodies against CD2, CD3, CD16, CD19, CD20, CD56, CD66b, CD123, glycophorin A and dextran-coated magnetic particles.…”

Section: Discussionmentioning

confidence: 99%

An <em>In vitro</em> Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Erbel

Rupp

Helmes

et al. 2013

JoVE

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Isolation of Human Monocyte Populations

Cited by 54 publications

References 7 publications

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin

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

An <em>In vitro</em> Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

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