Computational protocol: The TRPM2 ion channel is required for sensitivity to warmth

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[…] Cells were loaded with 5 μM fura-2, AM (Invitrogen) with 0.02% (v/v) pluronic acid (Invitrogen) in growth medium for 30 min. After loading, coverslips were put in an imaging chamber and transferred to a Nikon Eclipse Ti-E inverted microscope. Cells were continuously perfused with extracellular solution and were illuminated with a monochromator alternating between 340 and 380 nm (OptoScan; Cairn Research), controlled by WinFluor 3.2 software (Dr. John Dempster, University of Strathclyde, Glasgow, UK). Emission was collected at 510 nm and the resulting pairs of images were acquired every two seconds with a 100 ms exposure time using an iXon 897 EM-CCD camera (Andor Technology, Belfast, United Kingdom). Image time series were converted to .tiff and processed with ImageJ software. Images of the background fluorescence intensity were obtained for both wavelengths and subtracted from the respective image stack before calculating the F340/380 ratio images. A minority of neurons (<10%) exhibited an unstable F340/380 baseline in the absence of any applied stimulus, usually caused by poor dye loading but in some cases apparently due to low-frequency repetitive firing even in the absence of any treatment, and were removed from analysis. In experiments to identify neurons responding to known TRP channel agonists (), we found that PS caused a very slow increase in F340/380 ratio in some neurons, clearly distinguishable from the rapid elevation in [Ca]i seen in TRPM3-expressing DRG neurons. This slow response can probably be attributed to an off-target effect of PS as it was also seen in autonomic neurons which do not appear to express TRPM3 (). A positive response to all agonists was therefore defined from the rate of increase of [Ca]i following agonist application, as an increase of F340/380 ratio, between two consecutive time points following application of agonist, which exceeds the mean + 3.09 SD (cumulative probability value of 99.9%) of all such differences in the absence of any agonist. A heat-sensitive neuron is defined as a neuron with a peak increase in F340/380 during a heat stimulus larger than the mean + 3.09 SD of the peak increase in F340/380 of the glial cells in the same experiment (see ). The thermal threshold of a heat-responsive neuron (see ) was defined as the temperature when the increase in F340/380 ratio between two consecutive time points is larger than the mean + 3.09 SD of the increase in F340/380 of the glial cells between two consecutive time points in the same experiment. For MAH and PC12 cell cultures, where no glial cells were present, we used the value of mean + 3.09 SD obtained from glial cells in similar experiments on neuronal cultures. shows an example series of calcium images. [...] MAH cells were trypsinized and collected as a cell pellet prior to lysis. RNA extraction was performed with the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. 2 samples from MAH cells grown in growth medium and 2 samples from MAH cells grown in differentiation medium were sent to Oxford Gene Technology to complete the rest of the steps for RNA-sequencing. Sequencing libraries were prepared with the Illumina TruSeq RNA Sample Prep Kit v2. A total of 4 samples (two cold-sensitive MAH cells and two cold-insensitive MAH cells) were sequenced on 2 lanes on the Illumina HiSeq2000 platform using TruSeq v3 chemistry. All sequences were paired-end and performed over 100 cycles. Read files (Fastq) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were processed through the Tuxedo suite. Reads were mapped to their location to the appropriate Illumina iGenomes build using Bowtie version 2.02. Splice junctions were identified using TopHat, version v2.0.9. Cufflinks version 2.1.1 was used to perform transcript assembly, abundance estimation and differential expression and regulation for the samples. Visualisation of differential expression results were performed with CummeRbund. RNA-Seq alignment metrics were generated with Picard. [...] Following hybridization coverslips were washed in 2 x SSC and then 0.2 x SSC at 47 °C for 30 min for each solution. Coverslips were then washed twice with KTBT solution at room temperature for 5 min for each washing. 25% normal goat serum was then used for blocking cells for 1 h at room temperature. Coverslips were then incubated in 25% normal goat serum containing pre-absorbed anti-digoxygenin antibody coupled to alkaline phosphatase for 2 h at room temperature, followed by washing 3x in KTBT for 15 min each wash, and then twice in alkaline phosphatase buffer at room temperature for 10 min each wash. Coverslips were then developed in alkaline phosphatase buffer containing 337.5 μg/ml NBT and 175 μg/ml BCIP in the dark for 8 h before being washed in KTBT, fixed in 4% PFA for 10 min, washed in PBS, and then mounted in SlowFade Gold Antifade Mountant with DAPI. DIC transmitted-light images were acquired through a Plan Fluor 10x Ph1 DLL objective with a DS-Qi2 monochrome camera on a Nikon Eclipse Ti-E inverted microscope. A GFPHQ filter was used to enhance the dark purple color. The images were rotated, cropped, and resized with ImageJ to be aligned with the images obtained in calcium imaging. [...] All data are expressed as means ± s.e.m. Analyses were performed with GraphPad Prism version 6.01 or SigmaPlot 11.0. The particular statistical test used is stated in the text or Fig legends. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Databases BCIP
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Calcium