D glucose

Immortalized mouse brain endothelial cells (ECs), bEnd.3 cells (ATCC, Manassas, VA, USA), were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 4500 mg/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate, 1.5 g/L sodium bicarbonate; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Mediatech Inc., Manassas, VA, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Welgene). bEnd.3 cells were incubated in a humidified incubator containing 5% CO₂ at 37 °C until confluence.

To evaluate cell mitochondrial stress, a Seahorse XF2 analyzer (Agilent, 102342-100) and XF Cell Mito Stress Test Kit (Agilent, 103015-100) were used. The drugs used in this assay were as follows: oligomycin (2.5 µM), FCCP (2 µM) and rotenone/antimycin A (0.5 µM).
At 2 h postinfection, the media of infected BMDMs (5 × 10⁵) was replaced with XF RPMI base medium (Agilent, 103576-100) containing 300 mg/L L-glutamine (Thermo, 25030081) and 2000 mg/L D-glucose (Welgene, LS001-01) and incubated in the absence of CO₂ for 1 h. After 1 h of incubation, a cell mito stress test was conducted.

The bEnd.3 cell line, immortalized mouse brain endothelial cells, was obtained from American Type Culture Collection (Manassas, VA, USA). bEnd.3 cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium with 4500 mg/L d-glucose, 110 mg/L sodium pyruvate, 1.5 g/L sodium bicarbonate, and l-glutamine; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Mediatech Inc., Manassas, VA, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Welgene, Korea). bEnd.3 cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% air. All experiments were carried out when the density of cells was 90–100%.