After the baseline was established at 35��C, DRG neurons were cooled in a ramp-shaped manner to 15��C over a period of 180?s, followed by heating back to 35��C within 1?s. After 60?s, 1?nM P-CTX-1 was applied for 360?s, during which a second cold stimulus was delivered. For quantitative comparison of cold sensitization, the maximum increase in intracellular Calcium after cold stimulation, relative to the BLZ945
10 baseline reads prior to the cold stimulus, was determined for control and ciguatoxin-treated cold responses. Neurons responding to the control cold response with an increase in intracellular calcium of at least 20?nM were classified as cold sensitive (34/204 neurons from wt and 28/406 neurons from TRPA1?/? mice), while neurons with an increase in [Ca2+] of <20?nM were classified as cold insensitive. Cold sensitization was defined as cold responses from previously cold-insensitive neurons that were at least five times larger after treatment with P-CTX-1 compared to control (67/170 neurons sensitized to cold in wt neurons and 25/378 sensitized to cold in TRPA1?/? neurons). Cos-1, HEK293 and ND7/23 cells (all from American Tissue Culture Collection, Manassas, VA, USA) were routinely maintained in DMEM containing 10% fetal bovine serum, 2?mM L-glutamine, pyridoxine and 110?mg/ml sodium pyruvate. Cells were split every 3�C6 days in a ratio of 1:5 using 0.25% trypsin/EDTA. Cells were plated on T75 tissue culture flasks (Nunc) 24?h prior to transfection and transfected with plasmid DNA of hTRPV1 (J Davis, formerly GlaxoSmithKline, Harlow, UK), mTRPA1 (A Patapoutian, The Scripps Research Institute, La Jolla, CA, <a href="http://www.selleckchem.com/products/ldk378.html
">find more USA), rNav1.8 (C Nau, Department of Anesthesiology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany), hTRPA1 (OriGene Technologies, Rockville, MD, USA) and hNav1.7 (N Klugbauer and F Hofmann, Technische Universit?t Munich, Germany) using Fugene (Roche) Pictilisib research buy
or Nanofectin (Paa, Austria) according to manufacturer's instructions. Twenty-four hours after transfection, cells were plated on 96-well plates, 384-well plates or glass coverslips as required and used 24?h after plating. The effects of P-CTX-1 on C- and A-fibres were assessed using single fibre recordings from isolated rat and murine skin-saphenous nerve preparations as previously described (Zimmermann et al, 2009). The hairy skin of the dorsal hind paw and lower leg of male adult C57BL/6, TRPA1?/?, Nav1.8?/? mice was removed together with the saphenous nerve, secured to the bottom of an organ chamber with the epithelial side facing down and continuously perfused with carbogenated SIF. Single A- and C-fibres were isolated from split single fibres of the desheathed saphenous nerve placed in a separate recording chamber under paraffin oil immersion. The receptive fields of the single fibres were identified using mechanical probing.