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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function

Fig. 3

The impact of functional HCN and KCNQ/Kv7 channels on cell count, survival, and proliferation of primary microglia.*p < 0.05; **p < 0.01; ***p < 0.001 compared to controls. a The total number of viable cells was counted after blockade of the HCN channel with ZD7288 (10 μM and 30 μM, n.s.), and after blockade of KCNQ/Kv7 channel with XE-991 (10 μM and 30 μM). Data are shown as mean values per field of view (FOV). b Ratio of viable versus dead primary microglia subjected to ZD7288 and XE-991 as assessed by live/dead-assay (10 μM and 30 μM of each; F(4, 25) = 4.129, p = 0.011, ω = 0.375). Data are shown as a ratio of living cells normalized to control = 1 (absolute percentage of living cells of control = 93.5%). c Release of LDH was measured photometrically (LDH-assay) as a surrogate for cell death after treatment of microglia with ZD7288 or XE-991 (10 μM and 30 μM each). Lysed cells served as control (H(5) = 28.27, p < 0.0001; positive control is significant to all other conditions). d Proliferation rate was measured by Ki67 expression on RNA level by RT-qPCR after blockade of the HCN channel with ZD7288 and XE-991 (10 μM and 30 μM each; F(4,17) = 6.059, p = 0.003, ω = 0.48). e Proliferation rate after treatment with ZD7288 and XE-991 (10 μM and 30 μM each) was revealed by BrdU incorporation (F(4,19) = 3.319, p = 0.032, ω = 0.273). f Migration of microglia in the Boyden chamber assay under the influence of ZD7288 and XE-991(10 μM and 30 μM each; F(4, 47) = 4.846, p = 0.002, ω = 0.228). Data were blank-corrected and normalized to control. g Upper row: representative immunocytochemical images of the live/dead-assay. All cells regardless of viability stained by Hoechst (blue) and dead cells were identified by propidium iodide incorporation (red); scale bars = 100 μm. Lower row: representative images of the BrdU-proliferation assay are shown. Hoechst stains all cell nuclei blue, BrdU (green) identify proliferating cells. Scale bars = 50 μm

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