Foetal calf serum

PSI cl.1, B1OXI and CLAB cell lines were grown in advanced Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Grand Island, USA), supplemented with 5% foetal calf serum (Lonza, Basel, Switzerland), 2 mmol/L L-glutamine (Sigma), 100 U/mL penicillin (Sigma) and 1 mg/mL streptomycin (Fluka, Buchs, Switzerland), at 37 °C in a humidified atmosphere of 5% CO₂. To form monolayers, 96-well microplates were seeded with approximately 5.0 × 10⁵ cells/mL PSI cl.1 and CLAB, and incubated for 24 h to reach confluence. Thereafter, the cultures were washed three times with phosphate-buffered saline (PBS) and cultivated in antibiotic free DMEM for the cell adhesion and invasion assays.

Fibroblasts from the dominant ACO2 P15 patient (c.1253-1254insA) and the recessive P51 patient (c.36 + 5del; c.719G>C), as well as from an ICRD patient with biallelic ACO2 variants (c.487G>A; c.2048G>A), were generated from skin biopsies and compared throughout the study to two wild-type fibroblast cell lines. Cells were cultivated in 2/3 Dulbecco’s Minimum Essential Medium (DMEM, Gibco) supplemented with 1/3 AmnioMAX (Gibco), 10% foetal calf serum (Lonza) and 1% Penicillin-Streptomycin-Amphotericin B (Lonza).

B16-F10 and B16-OVA murine melanoma cells were cultured at 37 °C under 5% CO2 in DMEM high glucose with GlutaMax-1 (Lonza) supplemented with 10% (v/v) foetal calf serum (Lonza), 1% penicillin, streptomycin, amphotericin B (Gibco), 4 mmol/l HEPES (Gibco) and 1 mmol/l sodium pyruvate (Gibco). B16F10 cells (ATCC CRL-6475) were obtained from American Type Culture Collection. B16-OVA cells were provided by J.D. Rosenblatt. All cells were routinely tested for Mycoplasma contamination using Mycoalert Mycoplasma Detection Kit (Lonza) and were found to be negative. To induce tumour formation, 2 × 10⁵ B16-F10 or B16-OVA cancer cells were injected subcutaneously into mice. After one week, tumour size were measured daily with an electronic calliper. According to our institutional ethical board, animals were killed by cervical dislocation, after have been anesthetised, when the maximum tumour size (2000 mm³) was achieved or when a necrosis superior to 2 mm was observed. Alternatively, 2 × 10⁵ B16-F10 or B16-OVA cells were injected intravenously into mice. Lung tumour foci were counted after 13 days by researchers ‘blinded’ to sample identity. All tumour growth experiments were approved by the Burgundy University Animal Experiments Ethics Committee (approved protocol #2212).

The U2OS cell line was cultured at 37 °C in DMEM (Dulbecco’s Modified Eagle Medium; Lonza) supplemented with 10% foetal calf serum (Lonza), 4 mM glutamine (Sigma-Aldrich) and 1x antibiotic (Sigma-Aldrich).

TEC 1A3 cells (a gift from Dr. Garchon, Inserm U580, Paris) were maintained in RPMI 1640 medium (Lonza) supplemented with 10% foetal calf serum (Lonza) and 1% penicillin/streptomycin. Previous research studying the AIRE promoter has been carried out using this cell line [11 (link)].

ASPC1, Mia PaCa-2 and PL45 were acquired from ATCC. BxPC3 are a kind gift from Dr. A. Frampton (Imperial College, London, UK). PaTuS and PaTuT were a kind gift from Dr. I. van Die (Amsterdam UMC, The Netherlands). Cell lines were tested for their authentication by STR-PCR, performed by BaseClear (Leiden, The Netherlands), previous to the start of the project. Additionally, all cell lines were routinely tested for Mycoplasma using PCR. All the cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% Foetal Calf Serum (Lonza), 2 mM l-glutamine (Gibco) and 1000 U/mL Penicillin-Streptomycin (Gibco).

L. infantum promastigotes (MCAN/ES/98/LLM-722, JPC strain) were grown in NNN medium and complete RPMI medium (RPMI1640, Gibco, Paisley, UK) supplemented with 100 UI/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-glutamine, 5×10⁻⁵ M 2-mercaptoethanol and 10% heat inactivated foetal calf serum (Lonza, Spain) as liquid phase for 2 weeks.