The voltage\gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain. treated microglia without PAP\1 (second/first peak ratio of 0.56??0.12; n = 249 cells from N = 3 experiments) and with PAP\1 (second/first peak ratio of 0.70??0.05; =?0.2220); n = 179 cells from N = 3 experiments). Statistical analysis applied was a paired t\Test. Error bars indicate mean? 0111:B4, Millipore Sigma) or 20?ng/ml IL\4 (Millipore Sigma), respectively, in reduced serum growth media with 2% FBS. 2.4. ICV Atipamezole injection of LPS LPS (O55:B5, Millipore) dissolved in phosphate\buffered saline (PBS) or PBS only as a vehicle was ICV administered stereotactically to 12\week\old male C57Bl/6J mice as previously described (Di Lucente et al., 2018). Briefly, a total volume of 2 l was injected per side into the lateral ventricles of anesthetized Atipamezole mice using a Hamilton syringe with a 27\gauge needle (Hamilton, Reno, NV). Injection sites were coordinated at ?1?mm posterior to the bregma, 1.3?mm lateral to the sagittal suture, and 2?mm in depth. The incision was surgically closed Atipamezole and mice were placed on an isothermal pad at 36C and continuously monitored following surgery until recovery. Twenty\four hours later, mice were euthanized, and the brain tissues were processed for isolation of CD11b?+?microglia for quantitative PCR (qPCR) analysis. 2.5. Middle cerebral artery occlusion Microglial Kv1.3, Kir2.1 and P2X4 expression was studied in brains of 16?week\old male or female CX3CR1+/GFP mice subjected to transient MCAO surgery (60?min occlusion) with 8?days of reperfusion as described previously (Chen et al., 2018). 2.6. Acute microglia isolation Microglia isolation using Miltenyi CD11b\conjugated magnetic microbeads was described previously (Chen et al., 2018). Briefly, brains were quickly dissociated enzymatically with a Neural Tissue Dissociation Kit (Miltenyi Biotec). Microglia were subsequently purified by magnetic\activated cell sorting (MACS) using anti\CD11b magnetic beads (Miltenyi Biotec). The whole process took about 60C90?min and isolated cells were immediately used for downstream applications without culturing or exposure to serum. 2.7. Electrophysiology Recordings from cultured and ex vivo acutely isolated microglia were performed on cells that were plated on poly\lysine\coated glass coverslips immediately after trypsinCEDTA detaching or isolation, respectively. All electrophysiological measurements started after cells were incubated at 37C for 10 min. Currents were recorded using the whole\cell configuration of the patch\clamp technique at room temperature with an EPC\10 HEKA amplifier and the HEKA PatchMaster 9 data acquisition software. External normal Ringer solution contained 160?mM NaCl, 4.5?mM KCl, 2?mM CaCl2, 1?mM MgCl2, 10?mM HEPES, pH 7.4, and 300?mOsm. Patch pipettes were pulled from soda lime glass (micro\hematocrit tubes, Kimble Chase, Rochester, NY) to resistances of 2C3 Ntn1 M when submerged in the bath solution and filled with an internal solution containing 160?mM KCl, 2?mM MgCl2, 10?mM HEPES, and 10?mM EGTA, pH 7.2, 300?mOsm. Kv1.3, Kir, P2X4, and P2X7 currents were recorded in the voltage\clamp mode. K+ currents were elicited with 200\ms voltage ramps from ?120 to +40?mV at a frequency of 0.1 Hz. Inward rectifier (Kir) currents were measured as peak inward currents at ?120?mV. Kv1.3 currents were measured as PAP\1\ or ShK\223\sensitive delayed rectifier outward currents elicited at voltages positive to ?40?mV from the same cell. Kir and Kv1.3 current densities were determined by dividing their current amplitudes in picoamperes (pA) at ?120?mV (Kir) or?+?40?mV (Kv1.3) by the cell capacitance measured in picofarads (pF). P2X currents were elicited by an application of either a 2\s pulse of ATP or a 3\s pulse of BzATP using triple\barreled theta glass and a Perfusion Fast\Step SF\77 System (Warner Instruments, Hamden, CT) at a holding potential of ?60?mV. P2X current densities were determined by dividing their current amplitudes in picoamperes (pA) by the cell capacitance measured in picofarads (pF). Cell capacitance was directly measured by a lock\in function built in the HEKA PatchMaster 9 data acquisition software. Resting membrane potential (RMP) and membrane depolarization induced by current ramp steps or ATP were measured in current\clamp mode. Seal and access resistance, as well as cell capacitance, a direct measurement of cell surface area, were monitored.