Sodium pyruvate solution

Spleen lymphocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS (Biochrom GmbH), 1 mM HEPES Buffer Solution (Life Technologies), 25 mM sodium pyruvate solution (Sigma), 0.5 mM 2-mercaptoethanol (Bio-Rad), MEM Non-essential Amino Acid Solution 1 × (Sigma), 100 U/ml Penicillin and 100 U/μg/ml Streptomycin (Life Technologies).

Patient and control cultured skin fibroblasts were grown in minimum essential medium (MEM) (Gibco) or Dulbecco's Modified Eagle Medium supplemented with 10% foetal bovine serum (Gibco), 1× MEM vitamins (Sigma), 1× non-essential amino acids (Sigma), 50 U/ml penicillin, 50 μg/ml streptomycin (Sigma), 100 mm sodium pyruvate solution (Sigma), 0.05 mg/ml uridine aqueous solution and 2 mm L-glutamine (Sigma).

Human embryonic kidney (HEK) 293 cell line was obtained from ATCC and cultured in DMEM medium (Gibco, Invitrogen) supplemented with 10% FBS (ExCell Bio), 100 μg/ml streptomycin (Sangon Biotech), and 100 U/ml penicillin (Sangon Biotech) at 37 °C in a humidified incubator containing 5% CO₂. The mouse renal carcinoma cell line (Renca) was acquired from Cobioer Biosciences (Nanjing, China) and verified by an analysis certificate. The stable cell line hCAIX-Renca was established and stored in our laboratory^{29 (link)}. Renca or hCAIX-Renca was cultured in RPMI-1640 medium (Gibco, Invitrogen) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate solution (Sigma), 1× MEM non-essential amino acid solution (Sigma), and 2mM L-glutamine (Sigma).

Human Embryo Kidney (HEK) 293T (ATCC: CRL-11268) cells, BEK (Bovine Embryo Kidney) cells from Dr. M. Ferrari, Istituto Zooprofilattico Sperimentale, Brescia, Italy (BS CL-94), BEKcre, expressing “cre” recombinase [46 (link)] and Madin Darby bovine kidney (MDBK) cells (ATCC: CRL 6071) were maintained as suggested by the provider’s instructions. All cell lines were cultured in Eagle’s Minimal Essential Medium (EMEM, Gibco; Thermo Fisher Scientific, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco), 1mM of Sodium Pyruvate (Gibco), 2 mM of L-glutamine (Gibco), 100 IU/mL of penicillin (Gibco), 100 μg/mL of streptomycin (Sigma-Aldrich, Milano, Italy), and 0.25 μg/mL of amphotericin B (Gibco),—from here annotated as cEMEM and were incubated at 37 °C, 5% CO₂ in a humidified incubator. HEK-293T and BHK-21 (baby hamster kidney) cells (from the Cell Servicing Unit, The Pirbright Institute, UK) used in low bio-containment assays were maintained and cultured using Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat inactivated (HI) FBS (Life Science Production), 1% Sodium Pyruvate solution (Sigma-Aldrich, Milano, Italy) and 1% penicillin–streptomycin (10,000 U/mL; Life Technologies from Thermo Fisher Scientific)—from here annotated as cDMEM-10 and incubated at 37 °C, 5% CO₂.

BHK-21 cells were maintained in Dulbecco׳s Modified Eagle Medium (Gibco, 41985-039) supplemented with 10% fetal calf serum (Invitrogen), 2% tryptose phosphate broth (Sigma, T8159), 1% 1M HEPES solution (Sigma, H0887), 1% 100mM sodium pyruvate solution (Sigma, S8636) and 1% Penicillin-Streptomycin-Glutamine (Thermo Fisher Scientific, 10378016) throughout all experiments. 3 x 10⁵ BHK-21 cells per well were plated in 6-well plates and were left overnight at 37°C. The next day the sub confluent cells were infected at a multiplicity of infection (MOI) of 0.01 (assuming 6 x 10⁵ cells per well). For these infections LCMV Clone 13 WT, LCMV Clone 13 containing the GP34A->T mutation or LCMV Clone 13 containing the GP35V->A mutation was used. Three replicate wells were used for each condition. After infection 2ml of media was added to each well. After 6, 12, 24, 48 and 72 hours post infection 1ml of media was taken for further analysis and then 1ml of fresh media was added. At 96 hours post infection, all the media was taken.

A naturally transformed line of chicken macrophages named HTC cells [22 (link)] were cultured in Roswell Park Memorial Institute (RPMI) 1640 media (Thermo Fisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (Thermo Fisher Scientific), 1X antibiotic antimycotic solution (Sigma-Aldrich, St Louis, MO, USA), 1X sodium pyruvate solution (Sigma-Aldrich), gentamicin solution (Sigma-Aldrich), 10 mM glutamine solution (Thermo Fisher Scientific) at 37°C for 24–48 h in a humidified incubator containing 5% CO₂ as described earlier with minor modifications. The cells were cultured to semi-confluence followed by dissociation with Accumax (Sigma-Aldrich) to perform different assays.

The MDV-transformed HP8 lymphoblastoid cell lines (40 (link)) from a GA strain-induced tumor were grown at 38.5°C in 5% CO₂ in RPMI 1640 medium (Life technologies) containing 10% fetal bovine serum, 10% tryptose phosphate broth, 1% sodium pyruvate solution (Sigma), and 100 units/ml of penicillin and streptomycin (Life Technologies).