Ags crl 1739

The human gastric adenocarcinoma cell line (AGS; CRL:1739™) and murine macrophage cell line (RAW264.7, TIB-71™) were supplied by the American-Type Culture Collection (ATCC^®, Manassas, VA, USA). penicillin-streptomycin, fetal bovine serum (FBS; 10%), trypsin EDTA (0.25%), and Kaighn’s Modification of Ham’s F-12 Medium (F-12K) were supplied by Gibco™ (Invitrogen, CA, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a colorimetric reagent sourced from Invitrogen™ (Thermo Fisher Scientific, Eugene, OR, USA) for the analysis of viability and cytotoxicity assays.
Roswell Park Memorial Institute (RPMI-1640) medium, 10% FBS, penicillin (100 IU/mL), streptomycin (100 mg/mL) solution, phosphate-buffered saline (PBS; pH = 7.4), and trypsin-EDTA 0.25% were obtained from Gibco™ (Invitrogen, CA, USA). Lipopolysaccharide (LPS), extracted from Escherichia coli, Indomethacin, and modified Griess reagent, was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dimethyl sulfoxide (DMSO) was provided by RCI Labscan (Bangkok, Thailand). All other reagents were of analytical or pharmaceutical grade.

One normal human gastric epithelial mucosa cell line (GES-1) was obtained from the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences (Beijing, China). Five GC cell lines AGS (CRL-1739) and SNU-1 (CRL-5971) were primary purchased from the American Type Culture Collection (Manassas, VA, USA); HGC-27(94042256) was from European Collection of Authenticated Cell Cultures (Public Health England; Porton Down, Salisbury, UK); MKN74 (JCRB0255) and MKN1 (JCRB0252) were from Japanese Collection of Research Bioresources Cell Bank (Ibaraki city, Osaka, Japan). All of these cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Gaithersburg, MD, USA) medium plus 10% Fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. AGS and HGC-27 cells were exposed to Ly294002 (10 μM, Sigma-Aldrich) or Rapamycin (100 nM) for 1 day.

Three human GC cell lines including MKN74 (JCRB0255, intermediate differential adenocarcinoma), NUGC-3 (JCRB0822, poorly differential adenocarcinoma) and MKN45 (JCRB0254, poorly differential adenocarcinoma) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). AGS (CRL-1739) were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in RPMI1640 medium supplemented with 10% foetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Japan) in a 37°C humidified incubator containing 5% CO₂.

Human GC epithelial cell lines AGS (CRL-1739) and HEK293 (CRL-1573) were obtained from the American Type Culture Collection. Another GC epithelial cell line MKN45 was kindly provided by Professor Min-Chuan Huang from the Graduate Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University. AGS and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, Marlborough, MA, USA) and MKN45 cells in Roswell Park Memorial Institute medium (HyClone) at 37°C in a humidified incubator under 5% CO₂. Culture medium was supplemented with 10% fetal bovine serum (HyClone), penicillin (100 U/L), and streptomycin (10 mg/L). The H. pylori strain (ATCC 43504) was obtained from the American Type Culture Collection. H. pylori was inoculated on Columbia agar containing 5% sheep blood (BD Biosciences, San Jose, CA, USA) and grown at 37°C in a microaerophilic chamber under 10% CO₂, 5% O₂, and 85% N₂. The study on H. pylori was approved by the Biosafety Committee of National Taiwan University. All procedures were conducted in compliance with the approved guidelines and regulations. AGS and MKN45 cells were infected with H. pylori at multiplicity of infection (MOI) of 30 and 90, respectively, for the indicated times after starvation in serum-free medium for 1.5 h.

The AGS (CRL-1739) and MKN45 (JCRB0254) human gastric epithelial cell lines were obtained from the American Type Culture Collection and the Japan Health Science Research Resource Bank, respectively. The cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen) at 37°C in a humidified 5% CO₂ atmosphere. Human primary stomach epithelial cells (HPSEC) were obtained from CellBiologics, USA. HPSEC were cultured in epithelial cell medium (M6621, CellBiologics) following the instructions from the provider. As indicated, the cells were treated with purified rJHP0290 or the same volume of protein storage buffer.