Fetal calf serum (fcs)

The adherent colon carcinoma cell line HT-29 (ATCC/LGC GmbH, Wesel, Germany) was cultivated in McCoy’s 5A medium (Gibco, Carlsbad, CA, USA) with either 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany) for magnetic accumulation or cell death kinetics, or 10% Panexin NTA (PAN-Biotech GmbH, Aidenbach, Germany) whenever analyses of DAMPs or cytokines were planned, to minimize interactions with the detection. In preceding experiments, we confirmed that HT-29 cells showed similar behavior, no matter if cultivated in medium containing FCS or Panexin. The monocytic cell line THP-1 (TIB-202; American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mM glutamine, 10% FCS (Biochrom AG), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco^®). All cells were cultured in a cell culture incubator (INCOmed, Memmert, Schwabach, Germany) at 37 °C, 5% CO₂ and 95% humidified air. Cells were regularly checked for mycoplasma contamination using PCR kit Venor^®GeM (Minerva Biolabs GmbH, Berlin, Germany). Before every experiment, count and viability was analyzed using MUSE^® Count & Viability assay kit in MUSE cell Analyzer (Merck-Millipore, Billerica, MA, USA). A cell viability above 95% was required to start an experiment.

Three human Ewing sarcoma cell lines, namely: RD-ES (HTB-166™; ATCC, Manassas, VA, USA); A-673 (CRL-1598™; ATCC); and TC-71 (ACC 516; Leibniz Institute DSMZ, Braunschweig, Germany) were employed for all experiments. Bone marrow-derived mesenchymal stem cells (MSC) isolated (local ethical approval number: 885/2021BO2) and propagated in culture as previously described [32 (link)], were used as a control group. The RD-ES cells were cultured and maintained in RPMI-1640 with L-glutamine (Gibco, Life Technologies, Waltham, MA, USA) media, supplemented with 15% (v/v) FCS (Biochrom Ltd., Cambridge, UK), and 1% (v/v) penicillin/streptomycin (Gibco, Life Technologies) while A-673 cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, Life Technologies) supplemented with 10% FCS (Biochrom Ltd.) and 1% (v/v) penicillin/streptomycin (Gibco, Life Technologies). Iscove’s MDM with L-glutamine (Gibco, Life Technologies) supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin (Gibco, Life Technologies) was used for TC-71 cells.

Extracted third molars were collected with patient’s informed consent from 18 to 30-year old adults from Charité Center for Dental, Oral, and Maxillary Medicine (Berlin, Germany). Teeth were kept in DMEM high glucose (PAA, Linz, Austria) containing 10% FCS (Biochrom, Germany) and 1% antibiotic-antimycotic solution (PAA, Linz, Austria) and stored at 4 °C for not longer than 24 hours. Human dental pulp cells are isolated under sterile conditions according to a modified protocol^{13 (link)}. Briefly, extracted specimens were wiped with ethanol to reduce contamination risk. To open the pulpal cavity, teeth were cracked mechanically. Pulp tissue was removed with forceps and placed into a PBS containing petri dish. The tissue was minced and washed twice with PBS to remove debris and blood. Enzymatic tissue digestion was performed with a collagenase (3 mg/ml)/dispase II (4 mg/ml) enzyme mix in PBS for 1 h at 37 °C. After filtering through a 70 μm cell strainer and washing with PBS by centrifugation (400 × g for 5 min), the cell pellet is resuspended in DMEM with 10% FCS and antibiotics. Isolated dental pulp cells were cultivated and expanded in a humidified atmosphere at 37 °C, 5% vol/vol CO2 in standard tissue culture flasks (Greiner Bio-One GmbH, Germany) in DMEM high glucose (PAA) containing 10% FCS (Biochrom, Germany) and 1% antibiotic-antimycotic solution.

In brief, HEK-293 cells were grown in Petri dishes in Dulbecco’s modified Eagle’s medium (Gibco Thermo Fisher, Waltham, MA, USA) supplemented with 10% FCS (Biochrom GmbH, Berlin Germany). Cells were routinely passaged twice a week and incubated at 37°C under 5% CO₂ growth conditions. tsA-201 cells were cultured at 37°C and 5% CO₂ in a 60-mm-diameter cell culture dish in 4-ml DMEM GlutaMAX medium (Biochrom GmbH, Berlin Germany) with 10% FCS (Biochrom GmbH, Berlin Germany) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were routinely passaged twice a week. HEK-293 and tsA-201 cells were transfected using standard calcium phosphate method (Koch et al. 2016 (link)). For whole-cell and single-channel recordings, HEK-293 cells were transfected with a 1:0.5:1.5 ratio of Ca_V1.2 (α1c#77, NM_001129843.2) or Ca_V2.2 (NM_000718.3), either human WT (NM_000723) or mutant Ca_Vβ-subunit and human Ca_Vα₂-δ₁-subunit (NM_000722.3). For co-immunoprecipitation experiments, tsA-201 cells were transfected with a 1:2 ratio of either human WT or mutant Ca_Vβ_1b_HA-subunit and human Flag-Gem.

Human SW480, SW620, HCT116 colon carcinoma and isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines were grown in RPMI medium (Gibco, Life technologies, Darmstadt, Germany), 10% FCS (Biochrom, Berlin,Germany). The colon carcinoma lines CaCo-2 and HT-29 were grown in DMEM (Gibco), 10% FCS (Biochrom). All lines were kept at 37 °C, 5% CO₂. Claudin-3-YFP stably transfected cells were selected with 0.5–1.5 mgml^-1 G418 (Gibco). The expression vector for Cldn-3-YFP is based on pEYFP-N1 [9 (link)]. The identity of all cell lines was confirmed by STR-genotyping (DSMZ, Braunschweig, Germany).

Human pancreas carcinoma AsP-1, BxPC-3, Capan-1 and Sk-Mel-5 melanoma cells were grown in RPMI medium (Gibco, Life technologies, Darmstadt, Germany), 10% FCS (Biochrom, Berlin, Germany). The human pancreas carcinoma HUP-T3, MIA PaCa-2, PA-TU-8902 and human colon carcinoma HT-29 cells were grown in DMEM (Gibco), 10% FCS (Biochrom). All lines were kept at 37 °C, 5% CO₂. The identity of all cell lines was confirmed by STR-genotyping (DSMZ, Braunschweig, Germany).