Cell culture, transfection, oxygen–glucose deprivation treatment, and co-culturing
The human acute myeloid leukaemia cell line, HL-60, was used as the neutrophil model. HL-60 cells were cultured in 1640 RPMI medium containing 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in an atmosphere of 5% CO2. HL-60 cells were transfected with a mixture of H19 siRNA, miR-29b antagomir or control, and Lipofectamine RNAiMAX (GenePharma); subsequently, they were incubated for 24 h in a humidified incubator for further analyses.
Human brain capillary endothelial cells (hCMEC/D3) were grown in EBM-2 medium supplemented with vascular endothelial growth factor, insulin-like growth factor-1, epidermal growth factor, basic FGF, hydrocortisone, ascorbate, penicillin-streptomycin, and 2.5% FCS. Subsequently, they were maintained in an incubator at 37°C and 5% CO2. Oxygen–glucose deprivation (OGD) and reoxygenation were used as in vitro models for replicating cerebral ischaemia. The hCMEC/D3 cells were kept in a glucose-free and hypoxic incubator chamber with a gas mixture of 94.5% N2, 0.5% O2, and 5% CO2 at 37 °C for 2.5 hours, followed by co-culturing with HL-60 cells for 24 h. The hCMEC/D3 cells were divided into five groups: Sham + control-HL-60 group; OGD + control-HL-60 group; OGD + miR-29b antagomir-HL-60 group (20 nM); OGD + H19 siRNA-HL-60 group (20 nM); and the OGD + miR-29b antagomir (20 nM) and H19 siRNA (20 nM) groups.
In the co-culture experiments, 10 × 105 hCMEC/D3 cells/well were co-cultivated with HL-60 cells treated with H19 siRNAs, miR-29b, or controls under non-contact conditions. Further, 10 × 105 HL-60 cells/well were seeded on the apical side of Transwell membranes (Corning, 0.4 µm pore size) and cultivated with supplemented medium. Moreover, hCMEC/D3 cells were seeded to the basal side and further cultivated as aforementioned in the static monoculture model. At 24 h after co-culture, the cells were separately collected for further analysis.