Sodium pyruvate

Murine macrophage J774A.1 cell line (ATCC^® TIB67™, Manassas, VA, USA) were maintained in a growth medium composed of Dulbecco’s Modified Eagle’s Medium (DMEM; BI, Kibbutz Beit Haemek, Israel), supplemented with 10% (v/v) heat inactivated fetal bovine serum (HI-FBS; Gibco, Paisley, UK), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), and 0.5% (v/v) sodium pyruvate (Sigma-Aldrich). Human myeloid leukemia cell line THP-1 (a kind gift from Prof. Gabriel Nussbaum, The Innate Immunity Laboratory, The Hebrew University-Hadassah School of Dental Medicine, Israel) were cultured in RPMI 1640 medium (ATCC^® 30-2001™) containing 10% (v/v) HI-FBS, 1% (v/v) penicillin-streptomycin and 1% (v/v) sodium pyruvate. Cells were maintained at 37 °C in a humidified 5% (v/v) CO₂ incubator.

Casein sodium salt and acid casein, both from bovine milk, acrylamide (AAm), N,N’- methylenebisacrylamide (MBAAm), ammonium persulfate (APS), N,N,N’,N’ tetramethylenediamine (TEMED), N-(3-aminopropyl)methacrylamide hydrochloride (APMA), lysozyme from chicken egg white (40,000 units/ mg protein), phosphate buffered saline (PBS), NaCl, KCl, NaH₂PO₄, Dulbecco’s modified eagle’s medium (DMEM) (D5796), bovine calf serum, penicillin-streptomycin solution, sodium pyruvate, NIH/3T3 fibroblasts (ATCC^® CRL-1658/Sigma 93061524), trypsin EDTA (ethylenediaminetetraacetic acid) solution, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid solution (MTT solvent), and methanol were all purchased from Sigma (St. Louis, MO, USA). NaHCO₃ was purchased from Panreac (Barcelona, Spain), and NaOH pellets (99%), from Merck (Darmstadt, Germany). Octiset^® was purchased from Schülke (Norderstedt, Germany), and polyhexanide (polyhexamethylene biguanide hydrochloride, PHMB) 94%, from Carbosynth (Berkshire, UK). Mueller–Hinton Agar and Mueller–Hinton Broth were purchased from Oxoid Ltd. (Hampshire, UK). Distilled and deionised (DD, 18 MΩcm, pH 7.7) water was obtained with a Millipore system (Millipore Merck, Darmstadt, Germany).

MB002 and MB004 were gifts from Y.J. Cho (Oregon Health and Science University, Portland, OR, United States). D556^{58 (link)} was a gift from D. Bigner (Duke School of Medicine). D556 and D283 (D. Bigner, ATCC) cell lines were maintained in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin (ThermoFisher Scientific). MB002 and MB004 cells were maintained in culture medium with 1:1 DMEM/F12 (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12) and Neurobasal™-A Medium supplemented with non-essential amino acids, Sodium Pyruvate, HEPES, GlutaMax, B27, EGF, FGF, Heparin, LIF, 10% fetal bovine serum (ATCC), and 100 U/mL penicillin and streptomycin (All from ThermoFisher Scientific). All cell lines were maintained at 37 °C with 5% CO₂ in a 95% humidified atmosphere. All established cell lines were verified with STR analysis (GRCF, Johns Hopkins). Antibodies used in this study are listed in Supplementary Data 10.

The TF-1 CD34⁺ erythromyeloid leukemia cell line (ATCC, Manassas, VA) was grown in RPMI 1640 medium with L-glutamine (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GemCell, Sacramento, CA), antibiotics (penicillin and streptomycin, at a concentration of 0.04 mg/mL each, Cellgro), glucose (4.5 g/mL, Cellgro), sodium pyruvate (1 mM, Cellgro) and HEPES (10 mM, Cellgro), and recombinant human granulocyte-macrophage-colony stimulating factor (rhGM-CSF, 2 ng/mL; eBioscience, San Diego, CA). The cells were maintained at a density between 1 and 5 × 10⁵ cells/mL.
The U-937 human promonocytic cell line (ATCC, CRL-1593.2) was grown in RPMI 1640 medium with L-glutamine supplemented with 10% heat-inactivated FBS, antibiotics (penicillin and streptomycin, at a concentration of 0.04 mg/mL each), glucose (4.5 g/mL), sodium pyruvate (1 mM), and HEPES (10 mM). The cells were maintained at a density between 1 and 5 × 10⁵ cells/mL.
The human T-cell line, Jurkat (ATCC, TIB-152), was grown in RPMI-1640 media with L-glutamine supplemented with 10% heat-inactivated FBS, sodium bicarbonate (0.05%, Cellgro, Herndon, VA), and antibiotics (penicillin, streptomycin, and kanamycin at 0.04 mg/mL each, Cellgro, Herndon, VA). The cells were maintained at a density between 1 × 10⁵ and 1 × 10⁶ cells/mL. All cells were maintained at 37°C in 5% CO₂ at 90% relative humidity.

The materials were sterilized by UV light for 0.5 h from both sides. The samples were inserted into polystyrene 24-well cell culture plates (TPP, Trasadingen, Switzerland) and were seeded with endothelial cells originating from bovine pulmonary artery (line CPAE ATCC CCL-209, Rockville, MA, USA) in a minimum essential Eagle medium (E-MEM) supplemented with 2 mM l-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential aminoacids and 1.5 g/L sodium bicarbonate (all chemicals from Sigma). In order to avoid potential adsorption of serum-derived proteins and masking the oligopeptidic ligands, fetal bovine serum (Sebak GmbH, Aidenbach, Germany) was added to the medium 5 h after cell seeding to a final concentration of 20 % in each well [23 (link)]. Each well contained 30,000 cells and 1.5 ml of the medium. The cells were cultured in a humidified atmosphere containing 5 % CO₂ in the air.

Vero E6 cells (originally ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete growth medium consisting of Dulbecco's modified Eagle medium with 10% fetal bovine serum (Hyclone) containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. The H1299-E3 cell line (H1299 originally from ATCC as CRL-5803) was propagated in growth medium consisting of complete Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids. Cells were passaged every second day. The H1299-E3 (H1299-ACE2, clone E3) cell line was derived from H1299 as described in our previous work^{1 ,17 (link)}. Cell lines were not authenticated. Cell lines tested negative for mycoplasma contamination.