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The influence of serum-supplemented culture media in a transwell migration assay

Omar Zaki SS, Kanesan L, Leong MYD, Vidyadaran S

Cell Biol Int, 2019 Oct;43(10):1201-1204.
PMID: 30811086 DOI: 10.1002/cbin.11122

Our work cautions against the use of serum-supplemented culture media in a transwell migration assay when using chemoattractants other than FBS. At 24 h, a 5% foetal bovine serum (FBS) gradient caused BV2 microglia to migrate toward the lower compartment of the transwell apparatus. Interestingly, FBS-supplemented media in the absence of a gradient also resulted in notable microglia migration. Serum can therefore confound the interpretation of a transwell migration assay when another chemoattractant is used.

Matched MeSH terms: Microglia/cytology*
Fulltext A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2 microglia

Haw RT, Tong CK, Yew A, Lee HC, Phillips JB, Vidyadaran S

J Neuroinflammation, 2014 Jul 30;11:134.
PMID: 25074682 DOI: 10.1186/1742-2094-11-134

BACKGROUND: We report a novel method of culturing microglia in three dimension (3D) using collagen as a substrate. By culturing microglia within a matrix, we aim to emulate the physical state of microglia embedded within parenchyma.
METHODS: BV2 microglia cell suspensions were prepared with type I collagen and cast into culture plates. To characterise the BV2 microglia cultured in 3D, the cultures were evaluated for their viability, cell morphology and response to lipopolysaccharide (LPS) activation. Conventional monolayer cultures (grown on uncoated and collagen-coated polystyrene) were set up concurrently for comparison.
RESULTS: BV2 microglia in 3D collagen matrices were viable at 48 hrs of culture and exhibit a ramified morphology with multiplanar cytoplasmic projections. Following stimulation with 1 μg/ml LPS, microglia cultured in 3D collagen gels increase their expression of nitric oxide (NO) and CD40, indicating their capacity to become activated within the matrix. Up to 97.8% of BV2 microglia grown in 3D cultures gained CD40 positivity in response to LPS, compared to approximately 60% of cells grown in a monolayer (Pmicroglia in 3D collagen gels also showed increased mRNA and protein expression of inflammatory cytokines IL-6, TNF-α and the chemoattractant MCP-1 following LPS stimulation.
CONCLUSIONS: In summary, BV2 microglia cultured in 3D collagen hydrogels exhibit multiplanar cytoplasmic projections and undergo a characteristic and robust activation response to LPS. This culture system is accessible to a wide range of analyses and provides a useful new in vitro tool for research into microglial activation.

Matched MeSH terms: Microglia/cytology*
Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia

Tan SW, Ramasamy R, Abdullah M, Vidyadaran S

Cell Immunol, 2011;271(2):205-9.
PMID: 21839427 DOI: 10.1016/j.cellimm.2011.07.012

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (pmicroglia viability.

Matched MeSH terms: Microglia/cytology
Bone marrow-derived mesenchymal stem cells modulate BV2 microglia responses to lipopolysaccharide

Ooi YY, Ramasamy R, Rahmat Z, Subramaiam H, Tan SW, Abdullah M, et al.

Int Immunopharmacol, 2010 Dec;10(12):1532-40.
PMID: 20850581 DOI: 10.1016/j.intimp.2010.09.001

The immunoregulatory properties of mesenchymal stem cells (MSC) have been demonstrated on a wide range of cells. Here, we describe the modulatory effects of mouse bone marrow-derived MSC on BV2 microglia proliferation rate, nitric oxide (NO) production and CD40 expression. Mouse bone marrow MSC were co-cultured with BV2 cells at various seeding density ratios and activated with lipopolysaccharide (LPS). We show that MSC exert an anti-proliferative effect on microglia and are potent producers of NO when stimulated by soluble factors released by LPS-activated BV2. MSC suppressed proliferation of both untreated and LPS-treated microglia in a dose-dependent manner, significantly reducing BV2 proliferation at seeding density ratios of 1:0.2 and 1:0.1 (pmicroglia proliferation. Additionally, MSC reduced the expression of the microglial co-stimulator molecule, CD40. Collectively, these regulatory effects of MSC on microglia offer insight into the potential moderating properties of MSC on inflammatory responses within the CNS.

Matched MeSH terms: Microglia/cytology
Fulltext Silver nanoparticles reduce brain inflammation and related neurotoxicity through induction of H2S-synthesizing enzymes

Gonzalez-Carter DA, Leo BF, Ruenraroengsak P, Chen S, Goode AE, Theodorou IG, et al.

Sci Rep, 2017 03 02;7:42871.
PMID: 28251989 DOI: 10.1038/srep42871

Silver nanoparticles (AgNP) are known to penetrate into the brain and cause neuronal death. However, there is a paucity in studies examining the effect of AgNP on the resident immune cells of the brain, microglia. Given microglia are implicated in neurodegenerative disorders such as Parkinson's disease (PD), it is important to examine how AgNPs affect microglial inflammation to fully assess AgNP neurotoxicity. In addition, understanding AgNP processing by microglia will allow better prediction of their long term bioreactivity. In the present study, the in vitro uptake and intracellular transformation of citrate-capped AgNPs by microglia, as well as their effects on microglial inflammation and related neurotoxicity were examined. Analytical microscopy demonstrated internalization and dissolution of AgNPs within microglia and formation of non-reactive silver sulphide (Ag2S) on the surface of AgNPs. Furthermore, AgNP-treatment up-regulated microglial expression of the hydrogen sulphide (H2S)-synthesizing enzyme cystathionine-γ-lyase (CSE). In addition, AgNPs showed significant anti-inflammatory effects, reducing lipopolysaccharide (LPS)-stimulated ROS, nitric oxide and TNFα production, which translated into reduced microglial toxicity towards dopaminergic neurons. Hence, the present results indicate that intracellular Ag2S formation, resulting from CSE-mediated H2S production in microglia, sequesters Ag+ ions released from AgNPs, significantly limiting their toxicity, concomitantly reducing microglial inflammation and related neurotoxicity.

Matched MeSH terms: Microglia/cytology*