A Rapid Microplate-Based Fluorometric Assay for Phagocytosis

Uff, C. R.; Pockley, A. Graham; Rw, Phillips

doi:10.3109/08820139309063419

Cited by 11 publications

(6 citation statements)

References 4 publications

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“…The fluorescence plate reader assay was conducted as explained in a previous study [29]. Plates were read on a Tecan Infinite M200 plate reader at an excitation wavelength of 643 nm and an emission wavelength of 665 nm.…”

Section: Methodsmentioning

confidence: 99%

The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses

Liu

et al. 2014

J Neuroinflammation

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BackgroundInsufficient clearance by microglial cells, prevalent in several neurological conditions and diseases, is intricately intertwined with MFG-E8 expression and inflammatory responses. Electromagnetic field (EMF) exposure can elicit the pro-inflammatory activation and may also trigger an alteration of the clearance function in microglial cells. Curcumin has important roles in the anti-inflammatory and phagocytic process. Here, we evaluated the ability of curcumin to ameliorate the phagocytic ability of EMF-exposed microglial cells (N9 cells) and documented relative pathways.MethodsN9 cells were pretreated with or without recombinant murine MFG-E8 (rmMFG-E8), curcumin and an antibody of toll-like receptor 4 (anti-TLR4), and subsequently treated with EMF or a sham exposure. Their phagocytic ability was evaluated using phosphatidylserine-containing fluorescent bioparticles. The pro-inflammatory activation of microglia was assessed via CD11b immunoreactivity and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and nitric oxide (NO) via the enzyme-linked immunosorbent assay or the Griess test. We evaluated the ability of curcumin to ameliorate the phagocytic ability of EMF-exposed N9 cells, including checking the expression of MFG-E8, αvβ3 integrin, TLR4, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) using Western blotting.ResultsEMF exposure dramatically enhanced the expression of CD11b and depressed the phagocytic ability of N9 cells. rmMFG-E8 could clearly ameliorate the phagocytic ability of N9 cells after EMF exposure. We also found that EMF exposure significantly increased the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and the production of NO; however, these increases were efficiently chilled by the addition of curcumin to the culture medium. This reduction led to the amelioration of the phagocytic ability of EMF-exposed N9 cells. Western blot analysis revealed that curcumin and naloxone restored the expression of MFG-E8 but had no effect on TLR4 and cytosolic STAT3. Moreover, curcumin significantly reduced the expression of NF-κB p65 in nuclei and phospho-STAT3 (p-STAT3) in cytosols and nuclei.ConclusionsThis study indicates that curcumin ameliorates the depressed MFG-E8 expression and the attenuated phagocytic ability of EMF-exposed N9 cells, which is attributable to the inhibition of the pro-inflammatory response through the NF-κB and STAT3 pathways.

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Section: Methodsmentioning

confidence: 99%

The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses

Liu

et al. 2014

J Neuroinflammation

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“…Determination of macrophage phagocytosis was performed as described and modified previously (Uff et aL, 1993;Lehner et al, 2002). Briefly, 3.0 x 10 4 microglial well were plated in 96-well microtiter plates (Cellstar, Greiner, Germany) and allowed to adhere for at least 3 b.…”

Section: Phagocytosis Assaymentioning

confidence: 99%

CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-α release from lipopolysaccharide-treated cells and mice

Ciallella

Saporito

Lund

et al. 2005

European Journal of Pharmacology

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CEP 11004, a mixed lineage kinase (MLK) inhibitor, was examined for its effects on tumor necrosis factor alpha (TNF a) production in human THP 1 monocytes, mouse BV 2 microglia, and C57Bl/6 mice. CEP 11004 inhibited TNF a secretion up to 90% in THP 1 cells incubated with 3 Ag/ml lipopolysaccharide, with an IC 50 of 137 T 14 nM. CEP 11004 also inhibited TNF a production in lipopolysaccharide stimulated microglial cells, but did not inhibit the initial increase in TNF a mRNA expression as measured by real time polymerase chain reaction (PCR). The mitogen activated protein kinases (MAPKs) phospho c jun N terminal kinase (JNK), phospho p38, and phospho MAPK kinase 4 (MKK4) levels were increased in THP 1 cells following lipopolysaccharide treatment, and were reduced by CEP 11004 treatment. For in vivo studies, CEP 11004 was injected 2 h prior to lipopolysaccharide (20 mg/kg) administration. CEP 11004 significantly inhibited TNF a production at doses of 1 10 mg/kg as measured by enzyme linked immunosorbent assay (ELISA). These results suggest that MLK blockade may be useful in inhibiting pro inflammatory cytokine production in a wide range of diseases. D

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“…Determination of macrophage phagocytosis was performed essentially as described (31), with the following modifications. BMDM (6.5 ϫ 10 4 /well) were plated in 96-well microtiter plates and allowed to adhere for at least 3 h. At different time points, tetramethylrhodamine-conjugated fluorescent E. coli particles were added to a final concentration of 5 g/ml.…”

Section: Determination Of Macrophage Phagocytosismentioning

confidence: 99%

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-Deficient Mice Show Increased Susceptibility to Listeria monocytogenes Infection

Lehner

Schwoebel

Kotlyarov

et al. 2002

The Journal of Immunology

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Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to d-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2−/− mice was associated with normal levels of IL-1β, IL-6, and IFN-γ, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1β, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2−/− mice displayed diminished IFN-γ synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-γ production required for activation of antibacterial effector mechanisms.

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A Rapid Microplate-Based Fluorometric Assay for Phagocytosis

Cited by 11 publications

References 4 publications

The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses

The amelioration of phagocytic ability in microglial cells by curcumin through the inhibition of EMF-induced pro-inflammatory responses

CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-α release from lipopolysaccharide-treated cells and mice

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-Deficient Mice Show Increased Susceptibility to Listeria monocytogenes Infection

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