A Real-Time Assay for Neutrophil Chemotaxis

Cano, Patricia; Vargas, Amandine; Lavoie, Jean‐Pierre

doi:10.2144/000114416

Cited by 17 publications

(10 citation statements)

References 36 publications

(31 reference statements)

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“…The number of cells migrated from the upper to the lower chamber was determined real time for 24 h with 15-min intervals after drug treatment. The change in the cell migration rate was expressed as cell index (CI) ( Bird and Kirstein, 2009 ; Cano et al, 2016 ; Selli et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages

Bariş

Efe

Gümüştekin

et al. 2021

Front. Mol. Biosci.

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The cholinergic anti-inflammatory pathway plays an important role in controlling inflammation. This study investigated the effects of varenicline, an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, on inflammatory cytokine levels, cell proliferation, and migration rates in a lipopolysaccharide (LPS)-induced inflammation model in RAW 264.7 murine macrophage cell lines. The cells were treated with increasing concentrations of varenicline, followed by LPS incubation for 24 h. Prior to receptor-mediated events, anti-inflammatory effects of varenicline on different cytokines and chemokines were investigated using a cytokine array. Nicotinic AChR–mediated effects of varenicline were investigated by using a non-selective nAChR antagonist mecamylamine hydrochloride and a selective α7nAChR antagonist methyllycaconitine citrate. TNFα, IL-1β, and IL-6 levels were determined by the ELISA test in cell media 24 h after LPS administration and compared with those of dexamethasone. The rates of cellular proliferation and migration were monitored for 24 h after drug treatment using a real-time cell analysis system. Varenicline decreased LPS-induced cytokines and chemokines including TNFα, IL-6, and IL-1β via α7nAChRs to a similar level that observed with dexamethasone. Varenicline treatment decreased LPS-induced cell proliferation, without any nAChR involvement. On the other hand, the LPS-induced cell migration rate decreased with varenicline via α7nAChR. Our data suggest that varenicline inhibits LPS-induced inflammatory response by activating α7nAChRs within the cholinergic anti-inflammatory pathway, reducing the cytokine levels and cell migration.

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Section: Methodsmentioning

confidence: 99%

Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages

Bariş

Efe

Gümüştekin

et al. 2021

Front. Mol. Biosci.

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“…The chemotaxis assay was carried out as described elsewhere 66 . Briefly, cell invasion and migration plates (CIM-Plate 16; Roche) were coated with 20 μg/ml fibronectin (Sigma) in PBS at 37 °C, 30 min, then washed twice with 50 μl PBS.…”

Section: Methodsmentioning

confidence: 99%

The in vitro host cell immune response to bovine-adapted Staphylococcus aureus varies according to bacterial lineage

Murphy

Niedziela

Leonard

et al. 2019

Sci Rep

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Mastitis is the most economically important disease affecting dairy cattle worldwide. Staphylococcus aureus is a highly prevalent cause of mastitis, causing infections ranging from sub-clinical to gangrenous. However, the interaction between the genotype of the infecting strain of S . aureus and the host response remains largely uncharacterised. To better understand the variation in presentation and outcomes of S . aureus -mediated bovine mastitis, we studied the interaction of a panel of mastitis isolates from several prominent bovine-associated lineages with bovine mammary epithelial cells (bMEC) and neutrophils. Significant differences in immune gene expression by infected primary or immortalised bMEC, or their elaboration of neutrophil chemoattractants, were observed and were dependent on the lineage of the infecting strain. Differences were also apparent in the invasiveness of S . aureus strains and their ability to survive killing by neutrophils. Our results demonstrate that a range of immune responses occur, suggesting the importance of S . aureus strain in dictating mastitis disease course. S . aureus lineages may therefore have adopted differing strategies for exploitation of the intramammary niche. Consequently, improved diagnosis of infecting lineage may enable better prognosis for S . aureus mastitis and reduce morbidity and economic loss.

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“…It is noteworthy that the concentration affected only a basic test system parameter (number of migrating cells under control conditions) but not the assay performance under toxicant challenge. These results suggest that ECM-substitute materials hold high promise in high-throughput toxicology applications and now encourage the exploration of these materials in other tests, e.g., chemotaxis assays for T-cells and neutrophils (Lin and Butcher, 2006;Cano et al, 2016). In this regard, it is worth emphasizing that the ZT Fn10 material has demonstrated excellent cell compatibility and that its sole protein composition, self-assembly and molecular domain granularity is reminiscent of that of typical biological matrices.…”

Section: Discussionmentioning

confidence: 89%

Strategy to replace animal-derived ECM by a modular and highly defined matrix_suppl1

Nesterenko

2020

ALTEX

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Fetal bovine serum (FBS) is an example of an animal-derived product used commonly as a cell culture supplement. The unknown composition of FBS and its high batch-to-batch variation make it difficult to reproduce in vitro experiments between different laboratories (van der Valk et al., 2018). So far, serumfree media are in routine use only for a few cell types, such as LUHMES cells (Scholz et al., 2011) or induced pluripotent stem cells (iPSCs) (Hackland et al., 2017;Reichman et al., 2017;Sung et al., 2019), but no universal, chemically-defined, serum-free culture medium is available (van der Valk et al., 2018). The FCSfree database 1 was launched to increase the use of serum-free IntroductionWhile cell culture experiments have successfully replaced several animal studies, many in vitro methods still use animal-derived materials. The search for chemically-defined and non-animal sourced cell culture material has become a research priority in its own right. The goal is to make in vitro systems more reproducible (independent of lot effects) and to avoid ethically problematic procedures currently used to produce cell culture medium supplements, extracellular matrix, and analytical tools such as animal-derived antibodies.

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A Real-Time Assay for Neutrophil Chemotaxis

Cited by 17 publications

References 36 publications

Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages

Varenicline Prevents LPS-Induced Inflammatory Response via Nicotinic Acetylcholine Receptors in RAW 264.7 Macrophages

The in vitro host cell immune response to bovine-adapted Staphylococcus aureus varies according to bacterial lineage

Strategy to replace animal-derived ECM by a modular and highly defined matrix_suppl1

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