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Fig. 2 | Journal of Neuroinflammation

Fig. 2

From: Activation of neuronal N-methyl-d-aspartate receptor plays a pivotal role in Japanese encephalitis virus-induced neuronal cell damage

Fig. 2

Blockade of NMDAR by MK-801 prevents JEV-induced neuronal cell death in vitro. a Cell viability assay at indicated concentrations of MK-801. b The analysis of apoptotic markers caspase-3 and phosphatidylserine in primary mouse neuron/glia cells. The cells were either mock-infected or infected with JEV at an MOI of 1. Subsequently, cells were treated with indicated concentrations of MK-801 or DMSO after infection and every 24 h post-infection for 2 days. Caspase-3 protein levels (upper panel) and phosphatidylserine localization (lower panel) were determined by immunoblotting and flow cytometry, respectively. Protein levels were quantified by immunoblot scanning and normalized to the amount of GAPDH levels. c Immunofluorescence staining of neuronal nuclei marker NeuN (green) and apoptosis marker caspase-3 (red). Primary mouse neuron/glia cells were subjected to infection and treatment paradigm (10 μM concentrations of MK-801) as described in b, and cells were stained with corresponding antibodies. The right panel shows the quantification of caspase-3-positive cells. Scale bar, 400 μm. d Measurement of intracellular Ca2+ concentration. Primary mouse neuron/glia cells were subjected to infection and treatment paradigm (10 μM concentrations of MK-801) as described in b. Subsequently, cells were treated with Fluo 4/Am and collected for analysis with flow cytometry. Data are expressed as means ± SEM from three independent experiments. *P < 0.05, **P < 0.01

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