Ionomycin

Antibodies (Ab, see Supplementary Data 4) to mouse CD19, CD5, CD11b, CD43, 4-1BBL, IFNγ, IL1β, TGFβ, IL10, IL6, CD11b, and CD45 and their isotype-matched control Ab were purchased from Biolegend, eBioscience, BD Bioscience, and R&D Systems, unless specified. For IC cytokine staining, cells were stimulated with 50 ng/ml PMA (Tocris Bioscience) and 500 ng/ml ionomycin (Tocris Bioscience) for 1–2 h, followed by Golgi stop for 3–4 h using 10 μmol/l of Monensin or Brefeldin A (eBioscience); and then stained following manufacturer’s instruction for IC fixation and permeabilization (eBioscience). Concentration of antibody used in FACS staining was 1 μg per 10⁶ cells. Data were analyzed on FACS Canto II (BD) or CytoFLEX (Beckman Coulter, Inc.) using FlowJo software (Tree Star, Inc.) or CytExpert software (Beckman Coulter, Inc.).

Fura-2 fluorescence was measured in mouse primary colonic epithelial cells (n = 14 from three WT mice and n = 11 from three TRPV4-KO mice) and CCD 841 cells with a standard bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose at pH 7.4 (adjusted with NaOH) at 25°C. Results are presented as ratios of fluorescence intensities obtained from fura-2 emissions at 340 nm and 380 nm. GSK101 (Merck KGaA, Darmstadt, Germany), 8,9-EET methylestel (Cayman Chemical, Michigan, USA) [18 (link)], ionomycin and RN1734 (from Tocris Bioscience, Bristol, UK) [19 (link)] were used as TRPV4 agonists, a positive control and a TRPV4 antagonist, respectively. Clodronate-liposomes (Katayama Chemical, Japan, 1 μM) were used to inhibit VNUT [20 (link), 21 (link)]. F340/F380 and dose-response curves were calculated and acquired with an image processing system (IP-Lab, Scanalytics Inc., Rockville, MD) and ImageJ software (http://rsb.info.nih.gov/ij/). Changes in ratios (Δ) were calculated by subtracting the mean basal values from peak values.

HeLa cells (ATCC, Cat#CCL-2) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂. Cells were transfected with mCherry-CaM and mEGFP-CaMKIIα (or its mutant) using Lipofectamine-2000 for 24–48 h, and subjected to fluorescence lifetime imaging in a solution containing (in mM) 130 NaCl, 20 HEPES, 2 NaHCO₃, 25 D-glucose, 2.5 KCl, 1.25 NaH₂PO₄, 0.8 MgCl₂ and 1.8 mM CaCl₂ (pH 7.3). Cells were treated with 3 µM ionomycin (Tocris) and then 5 min later 10 mM EGTA (Sigma).

About 4x10⁶ THP-1 cells were seeded in T75 flasks and differentiated towards immature dendritic cells (iDC) over a 5-day culture in 20 mL serum-supplemented RPMI 1640 in the presence of 100 ng/mL hIL4 (R&D Systems, 204-IL-020/CF) and 100 ng/mL hGM-CSF (Sigma-Aldrich, #GF304). To allow full maturation towards mature dendritic cells (mDC), iDC were collected via centrifugation, resuspended in serum free RPMI 1640 media supplemented with 200ng/mL hIL4, 100ng/mL hGM-CSF, 20ng/mL hTNFα (Sigma-Aldrich, #GF314), and 200ng/mL ionomycin (Tocris Bioscience, 2092/1), and plated at density 10,000/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Cells were kept in culture for 1 day to allow differentiation towards mDC, before beginning the transfection with MessengerMax-formulated mRNA to induce expression of the E6-E7 fusion protein in the vaccine. The day following the transfection, human antigen specific E7_11-20 T cells (Charles River Laboratories, #ASTC-1099) were added to the 96-well plate at density 20,000 cells/well. ASTC and mDC cells were kept in coculture for an overnight. The plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions and read under the CTL Immunospot Analyzer (ImmunoSpot^®). CD209 monoclonal antibody eB-h209 (eBioscience™) was used to characterize DC differentiation via Flow Cytometry.