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A putative anti-inflammatory role for TRPM8 in IBS 109 5 antibody TRPM8 (1:200, Alomone: ACC-049) with either CGRP (1:400, Thermo Fisher, ABS026 ‐ 05 ‐ 02), CD103 (1:400, DAKO, R7188) and CD11C (1:200, Abcam, ab33483), CD64 (1:400, DAKO, R7129), CD68 (1:400, DAKO, M0876) and mast cell tryptase (1:400, DAKO, MC052) was applied overnight (4°C). Tissues were then washed and incubated (60 min, room temperature) with species-specific Alexa Fluor conjugated secondary antibodies (1:400, A-11001, A-21451, A-11036, Carlsbad, CA). A Leica DM4000 epifluorescence microscope was used to visualize TRPM8+ immunoreactivity (IR). Immunoreactive cells for each target were counted manually by two independent observers in each section and averaged over five fields of view – all taken at 40x magnification. Observers were blinded to the source of tissue, i.e. IBS subtype, as tissue was assigned a collection number until data analysis. Immunohistochemistry experiments were performed in biopsies from 28 IBS patients (5/28 obtained from the Maastricht cohort) and 7 controls (all obtained from the London cohort). No primary controls were performed to eliminate non-specific bind of secondary antibodies, and TRPM8 control antigen was used to ensure binding specificity ( Supplementary Figure S5.4 ). Cytokine experiments IBS biopsies were placed in 200μL Krebs solution (control), or in icilin (treatment) (1 µm, Tocris, product code: 1531) in 200 μL Krebs solution, and were maintained at 37 o C with 95% CO 2 /5% O 2 overnight (16-18 h) in 96 well plates. The supernatant was then removed and aliquots stored at −20°C. Quantification of cytokines the IL-1 β , IL-6, TNF- α , IL-10, and IL-8 was performed using a customized human cytokine/ chemokine/growth factor assay panel (Milliplex MAP Multiplex assay, Merck Millipore, product codeHCYTOMAG-60K), and analyzed on Luminex MAGPIX Instrument with xPONENT 4.3 (Luminex Corporation). Cytokine experiments were performed using sigmoid biopsies from 12 IBS patients (all obtained from the London cohort). Statistical analysis Statistical analysis was performed using SPSS 26 (IBM Corporation) or GraphPad Prism, V.5.02. (GraphPad Software, Inc). Statistical analysis for cell counting was performed using the Mann-Whitney test for unpaired data. For the comparison of mRNA expression between IBS patients and controls, a multivariable linear regression was performed correcting for age and gender. Wilcoxon signed-rank test was performed to compare sigmoid mRNA with proximal colon mRNA levels (within subjects). Relation to symptoms was also assessed using multivariate linear regression correcting for age and gender. For cytokine release assays, paired analysis with statistical significance using