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, 2002), using BAC RP24-78N24 (Children's Hospital Oakland Research Institute, BACPAC Resources Center) containing selleck chemicals llc the entire locus of TRPM8 gene plus 108.6 kb upstream and 52 kb downstream flanking regions. Eight different founders were identified. All experiments were performed on line CM1. F2 offspring were obtained to study germline transmission. Correct EYFP expression in DRG, TG, and spinal cord was confirmed in all lines obtained. Following enzymatic dissociation, labeled DRG neurons were purified by FACS using a FACSAria III (BD Biosciences). The quantity and purity of sorted cells were verified by fluorescent microscopy. Custom-designed TaqMan low-density array plates (96 wells) (Micro Fluidic Cards; Applied Biosystems) were used to quantify relative mRNA expression in TRPM8 neurons. Samples from fluorescent and nonfluorescent cells were collected and analyzed simultaneously, using the threshold PD-1/PD-L1 inhibitor 2 cycle relative quantification method. Electrophysiological and Ca2+-imaging experiments were performed on primary cultures of TRPM8-expressing DRG neurons of WT and TASK-3KO mice, following procedures described elsewhere by Madrid et?al. (2009). Whole-cell voltage and perforated current-clamp recordings were performed at a temperature of 32��C�C34��C with a MultiClamp 700B amplifier (Molecular Devices). Basic electrophysiological properties (input resistance, rheobase current, spike duration, inward rectification, and frequency-current Thalidomide relationships) and quantitative characteristics of the action potential were analyzed in current-clamp mode. For analysis of the pH-sensitive current (IpH), the currents obtained at pH 7.4 and pH 6.0 were digitally subtracted, and the resulting I�CV curves were fitted with a standard GHK current equation that included Na+ and K+ permeability terms. Behavioral testing of cold sensitivity in adult male mice followed the method of Brenner, based on the withdrawal latency to the application of a pellet of dry ice to the glass surface where the animals rest (Brenner et?al., 2012). 1-1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl (propan-1-one) (C23) was a kind gift of Merck. BCTC was purchased from Tocris, l-menthol was purchased from Scharlau Chemie, and cesium chloride and zinc chloride were from Sigma-Aldrich. Unless stated otherwise, data are reported as mean �� SEM. Statistical significance (p?