Fig. 2

Expression of NKCC1 in RBMECs after OGD. a Confocal image demonstrated that NKCC1 expression was localized in RBMECs. RBMECs were fixed and stained with anti-NKCC1 (red) and DAPI-stained nuclei (blue) in the control group, OGD conditions, OGD+Bum group, and OGD+Vit group. Images shown are representative of at least three independent experiments. b, d Flow cytometry analysis and the quantification of NKCC1-positive cell numbers in RBMECs. Cells were obtained at 24 h after OGD and were subjected to analysis using a flow cytometer after being immunostained with NKCC1 antibodies. Representative results from three to four tests of NKCC1-positive cells from each group are shown. c, e Fluorescence imaging of Cl⁻ via MQAE stained in RBMECs. The green fluorescence indicated the intracellular level of Cl⁻ in RBMECs and the fluorescence intensity of the controls was high while the RBMECs subjected to OGD were markedly decreased. These changes are readily reversible after treatment of OGD-exposed cells with bumetanide (100 mM; 24 h) or vitexin (100 mM; 24 h). Data are shown as the mean ± SEM; ***P < 0.005 compared to the OGD condition, n = 8~10 per group, scale bar = 100 μm, based on a one-way ANOVA