Fig. 4

Kv1.3 channels regulate microglial taxis and formation of F-actin complexes induced by LPS. a In the gap closure assay of microglial taxis, BV2 cells were grown to near confluence followed by placement of a uniform scratch using a 200-μm pipette tip. Ability of microglia to close this gap was assessed by measuring the percentage of gap closure over a 24-h period. Paired (0 and 24 h) representative images from each treatment group are shown (a, left), and the comparison of percentage gap closure (over 24 h) is shown (a, right) (six replicates per condition). b BV2 microglial transmigration across a transwell membrane (8-μm pore diameter) after exposure to control, ShK-223, ShK-186, LPS or LPS+ShK-223, or ShK-186, towards serum-containing medium (10% fetal bovine serum). Following 24 h of transmigration, cells were detached from undersurface of the insert (0.25% Trypsin), and cells that successfully migrated across the membrane were counted on a hemocytometer (n = 3, independent experiments). c Comparison of F-actin containing focal adhesion complexes in BV2 microglia following exposure to control, ShK-223, LPS, or LPS+ShK-223. Fixed and permeabilized BV2 cells were labeled with phalloidin-rhodamine to detect F-actin (left: immunofluorescence images). The number of focal complexes were counted per cell at ×40 magnification (right) and compared (>25 cells counted per condition). d DCFDA assay of ROS production by brain mononuclear cells isolated from C57BL/6 mice treated with PBS, LPS, ShK-223, or LPS+ShK-223 IP for four consecutive days (n = 3, mice/group). Cells were loaded with DCFDA for 30 min and assayed for ROS activity by flow cytometry. e Flow cytometric phagocytosis assay of fluorescent (PE) microbeads by brain mononuclear cells isolated from C57B6/L mice treated with PBS, LPS, ShK-223, or LPS+ShK-223, n = 3/group). Dotted line: fluorescence of cells not exposed to beads; gray histogram: PBS-treated; black histogram: LPS-treated. The proportions of all phagocytic cells and highly phagocytic cells were compared across treatment groups (*p < 0.05, **p < 0.01, ***p < 0.005)