Foetal bovine serum (fbs)

Human A375 cell line (CRL-1619) and RPMI7951 (HTB-66) were purchased from the American Type Culture Collection (Manassas, VA, USA). A375 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% foetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel) and 1% penicillin/streptomycin (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) in an incubator with 5% CO₂ at 37 °C. RPMI7951 cells were cultured in Minimum Essential Medium Eagle, with Earle’s salts and non-essential amino acids (MEM, Sigma Aldrich; Merck KGaA), supplemented with 10% foetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel), 1% penicillin/streptomycin (Sigma Aldrich; Merck KGaA, Darmstadt, Germany), 1 mM sodium pyruvate, and 2 mM L-glutamine (Sigma Aldrich; Merck KGaA, Darmstadt, Germany both). Appropriate medium supplemented with 2% charcoal stripped FBS was used for all procedures, and the effects of 1,25(OH)₂D₃ were examined.

The human normal lung epithelial cell (NL‐20) and lung cancer cell lines (NCI‐H1975 and NCI‐H1650) were purchased from American Type Culture Collection (ATCC). Normal lung cell NL‐20 was cultured in Ham's F12 medium with 1.5 g/L sodium bicarbonate, 2.7 g/L glucose, 2.0 mmol/L L‐glutamine, 0.1 mmol/L nonessential amino acids, 0.005 mg/ml insulin, 10 ng/mL epidermal growth factor, 0.001 mg/mL transferrin, 500 ng/mL hydrocortisone and 4% foetal bovine serum. NCI‐H1975 and NCI‐H1650 cells were cultured in RPMI‐1640 containing penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% foetal bovine serum (Biological Industries, Israel) and grown at 37°C in a humidified incubator containing 5% CO₂. All cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 2 months.

TMP (purity: >98%) was purchased from Aladdin (China). Lipopolysaccharide (LPS) and Alcian blue 8GX and Acridine orange were obtained from Sigma (United States). Foetal Bovine Serum (FBS) was purchased from Biological Industries (Israel). Albumin Borine V (BSA) was purchased from Solarbio (China). Calcein AM was purchased from Beyotime. Trizol was purchased from Invitrogen (United States).

H1975 lung adenocarcinoma cells, 16HBE cells, and BEAS-2B normal lung epithelial cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human circulating lung tumor cell line CTC-TJH-01 was previously established and characterized by our laboratory 15 (link), 16 (link). Briefly, CTC-TJH-01 cells were cultured in F12K medium supplemented with 10% foetal bovine serum (Biological Industries, Israel) and penicillin-streptomycin (Gibco Life Technologies, Carlsbad, CA, USA). H1975, 16HBE and BEAS-2B cells were cultured in RPMI-1640 medium (Corning Cellgro, USA) containing 10% FBS and penicillin-streptomycin. All cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

The GC tumour tissues and paired normal tissues were taken from patients who underwent surgical removal at the First Affiliated Hospital of Xi'an Jiaotong University. The Biomedical Ethics Committee of the Medical Department of Xi'an Jiaotong University gave their approve to this work. All participants involved in this study gave their informed consent.
All cell lines used (GES‐1, MKN‐28, BGC‐823, SGC‐7901, MKN‐45 and AGS) in this study were provided from Cell Bank (Genechem,). Before doing any research, the Cell Bank validated all of the cell lines and screened them for mycoplasma. Cells were incubated at 37°C with 5% CO2 using RPMI 1640 medium (Thermo Fisher Scientific, Inc.) added with 10% foetal bovine serum (Biological Industries).

PBMCs were isolated by density centrifugation (Ficoll-Hypaque, Almeco, Esbjerg, Denmark and Lymphoprep, Axis-Shield, Oslo, Norway), washed thrice in phosphate buffered saline (PBS without calcium and magnesium from Gibco, Invitrogen, Thermo Fischer Scientific, Waltham, MA), and re-suspended in Hams F12 growth media (Panum Institute, Copenhagen, Denmark), supplemented with 5% foetal bovine serum (Biological Industries, Beit HaEmek, Israel), 0.029% L-glutamin (Panum Institute, Copenhagen University, Denmark), non-essential amino acids and Penicillin/Streptomycin (Gibco, Invitrogen, Thermo Fischer Scientific, Waltham, MA). All experiments consisted of three 24 well plates with final concentrations of 10⁶ cells per well (NUNC, Roskilde, Denmark).