Fetal bovine serum (fbs)

An Indo-1 solution was prepared by mixing 50 μg Indo-1 AM (Biomol) with 115 μl FBS (Biochrom) + 25 μl DMSO (Sigma-Aldrich) and 25 μl Pluronic-F127 (AAT Bioquest). 0.6 × 10⁶ or 1 × 10⁶ cells were incubated in 1 ml RPMI (Thermo Fisher Scientific) + 1% FBS (Biochrom AG) + 15 μl Indo-1 solution at 37°C for 45 min. Cells were washed and used at a concentration of 10⁵ cells in 500 μl RPMI (Thermo Fisher Scientific) + 1% FBS (Biochrom AG) for the measurement. Basal calcium levels were acquired for 1 min. Subsequently, the cells were stimulated with 6 μl pervanadate solution (10 mM orthovanadate + 30% H₂O₂), thapsigargin (to a final concentration of 1 μM) or antimouse IgM (to a final concentration of 10 μg/ml), and NIP-BSA-biotin (to a final concentration of 20 ng/ml). If extracellular calcium needed to be removed, the cells were incubated in 0.5 mM EGTA (Fluka) containing medium 5 min before the measurement and for the duration of the experiment. Data were acquired on an LSR II flow cytometer (BD) at the Lighthouse Core Facility of the University Clinic Freiburg and the Max Planck Institute for Immunobiology and Epigenetics.

All cell lines, with exception of HUVECs, were from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCT-116 and HT-29 were kept at 37 °C, humidified 5% CO₂ incubators and cultured with McCoys medium supplemented with 10% fetal bovine serum (FBS) (Biochrom, Berlin, Germany) and 1% Pen/Strep (Gibco, Thermo Fischer Scientific, Waltham, MA, USA). SW-1463 cells were kept at 37 °C, humidified atmospheric-air incubators and cultured with Leibovitz’s L-15 medium supplemented with 10% FBS (Biochrom) and 1% Pen/Strep (Gibco). Primary, single-donor early passage HUVECs were kindly provided by Dr. Joanna Kalucka (Department of Biomedicine, Aarhus University, Aarhus, Denmark) and cultured at 37 °C, 5% CO₂ humidified incubators with Endopan 300SL medium (PAN Biotech, Aidenbach, Germany). Cells between passages five to twelve (HCT-116, HT-29, SW-1463) and passages two to three (HUVECs) were used for experiments. Normal colon epithelial cells (passages 1 and 2) were cultures with DMEM/F12 medium (ATCC) supplemented with 10% non-inactivated FBS (Biochrom), 10 ng/mL cholera toxin (Sigma), 0.005 mg/mL insulin (Gibco), 0.005 mg/mL transferrin (Gibco), 100 ng/mL hydrocortisone (Sigma) and 20 ng/mL human EGF (Gibco). The final DMSO concentration experienced by cells in vitro was ≤ 0.25%.

HEK293T cells, mouse embryonic fibroblasts (MEFs), and OG2 MEFs were cultured in low‐glucose (1,000 mg/l) Dulbecco's modified Eagle's medium (DMEM, Sigma‐Aldrich), supplemented with 10% fetal bovine serum (FBS, Biochrom), 2 mM l‐glutamine and 1× penicillin/streptomycin (Sigma‐Aldrich), 1% non‐essential amino acids (Sigma‐Aldrich), and 0.10 mM β‐mercaptoethanol. OG2‐MEFs were isolated as described previously 1. Mouse embryonic stem cells (mESCs) and iPSCs were maintained in high‐glucose (4,500 mg/l) DMEM (Sigma‐Aldrich), supplemented with 10% fetal bovine serum (Biochrom), 5% serum replacement (Gibco), 2 mM l‐glutamine, 1× penicillin/streptomycin (Sigma‐Aldrich), 1% non‐essential amino acids (Sigma‐Aldrich), 1% sodium pyruvate (Sigma‐Aldrich), 0.10 mM β‐mercaptoethanol (Gibco) with 1,000 units of leukemia inhibitory factor (LIF; prepared in house) on a feeder layer of gamma‐irradiated MEFs; experiments under feeder‐free conditions were performed using mESC medium and 2,000 units of LIF, as previously described 67.