Calhex231

For cytokine analysis, 80 × 10³ MDM per 200 µl cell culture media (RPMI-1640 containing 10% FCS) were cultured at 37 °C and 5% CO₂ in 96-well plates at least in duplicates. Twenty hours after stimulation with 0.1–100 EU/ml lipopolysaccharide (LPS-EB Ultrapure, Invivogen, San Diego, CA, USA) and or calcium chloride, supernatants were removed, stored at −80 °C and later analysed by ELISA for IL-1β, TNF and IL-6 (BD Biosciences Pharmingen, San Diego, CA, USA) following the instructions of the assay.
Inhibition of CaSR and Caspase 1 was done by 20 µM Calhex231 (#4387, Tocris Bioscience, Bristol, UK) and 10µM Z-YVAD-FMK (sc-3071, Santa Cruz Biotechnology, Dallas, Texas, USA), respectively. The DMSO-diluted antagonists were added prior to calcium chloride. DMSO in the same dilution served as control.

Monocytes were directly stimulated after isolation in either standard RPMI1640 cell culture medium (high [P_i], 5.6 mM Na₂HPO₄, Gibco, Lifetechnologies) or customized RPMI1640 containing 1 mM Na₂HPO₄ (low [P_i]) supplemented with 10% FBS (Gibco, Lifetechnologies). 3 × 10⁵ monocytes were seeded in 96-well plates and 100 ng/ml LPS (Invivogen) was used for monocyte “priming”. Inhibitors were pre-incubated for 30–60 min prior to stimulation.
CaSR (established in our group) and NLRP3-deficient (Invivogen) THP-1 cells were cultured in RPMI1640/10% FBS/1% penicillin and streptomycin (pen/strep) and selection antibiotics as indicated in the data sheet. Assays were performed in 24-well plates. 5 × 10⁵ cells/well were plated for differentiation in 50 ng/ml PMA (Tocris)-containing medium for 2 days before LPS-priming (100 ng/ml) and stimulation.
The following reagents and inhibitors were used for several cell culture experiments. Na₂HPO₄, BaCl₂, fetuin-A from FBS were purchased from Merck, CaCl₂, sodium phosphonoformate tribasic hexahydrate, from Sigma, YM254890 from Wako chemicals, Calhex231, NPS2143, Latrunculin A, Cytochalasin D from Tocris, Latrunculin B, PAF C-16, LLOMe from CaymanChemicals, MgSO₄ from AppliChem, ATP from Roche, DMSO from Serva, N-fMLP from abcam, and CA-074-Me from Selleck-Chem.