Roswell park memorial institute 1640 medium

The duodenal and colonic biopsy specimens were obtained from 4 of the 6 healthy dogs during endoscopy. Immediately after collection, the endoscopic biopsy specimens were washed 5 times with ice‐cold Roswell Park Memorial Institute‐1640 medium (Sigma‐Aldrich, St. Louis, MO) supplemented with l‐glutamine, 10% fetal bovine serum (Biowest, Nuaillé, France), and 1% penicillin and streptomycin (Wako Pure Chemical Industries, Ltd, Osaka, Japan). After washing, each sample was cut into 2 pieces and seeded in a 24‐well flat‐bottomed plate; 1 was cultured with medium alone as a control and the other was incubated with medium containing 10 ng/mL of canine recombinant IL‐1β (R&D Systems, Inc, Minneapolis, Minnesota) at 37°C for 24 hours under 5% CO₂. The concentration of IL‐1β was determined according to a previous study,11 which reflected inflammatory conditions in humans18 and a rabbit model of colitis.19 After incubation, the samples were immediately preserved in RNAlater Solution (Thermo Fisher Scientific, Waltham, Massachusetts) for RNA extraction.

Colon adenocarcinoma cells DLD-1 (Horizon Discovery) were cultured in a 1:1 mixture of Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich) and Ham's F-10 nutrient mix (Lonza), supplemented with 10% fetal calf serum (FCS; BioWest), and 1% penicillin/streptomycin (Sigma-Aldrich). Human embryonic kidney cells HEK-293T, osteosarcoma cells U2OS, hTERT-immortalized human fibroblasts VH10, SV40-transformed human fibroblasts C5RO, XP4PA (XPC-deficient) (19 (link)), CS1AN (CSB-deficient) (20 (link)), CS1AN complemented with YFP-CSB^del (21 (link)), and XRCC1-YFP expressing MRC-5 were cultured in a 1:1 mixture of Dulbecco's Modified Eagle medium (Lonza) and Ham's F-10, supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM UltraGlutamine (Lonza), and 0.1 mM MEM Eagle non-essential amino acid solution (Lonza). All cells were cultured at 37°C, in 20% O₂ and 5% CO₂, except for VH10 cells that were cultured at 3% O_2. To generate MRC-5 cells expressing XRCC1-YFP, XRCC1-YFP cDNA (a gift from Anna Campalans) (22 (link)) was cloned into pLenti-CMV-Puro-DEST (a gift from Eric Campeau & Paul Kaufman) (23 (link)). Following lentiviral transduction, MRC-5 cells stably expressing XRCC1-YFP were selected by puromycin and fluorescence-activated cell sorting.

About 67 GC tissues along with noncancerous tissues adjacent to them were collected from subjects reported to have GC at The Second Xiangya Hospital, Central South University, Changsha, during the period from March 2015 to March 2017. In accordance with the WHO guidelines, the patients were subjected to pathological diagnosis. Patient history was gathered assuring no chemotherapy and radiation was taken prior to submitting patients to surgery. The experimental protocol received sanction from the ethical committee of The Second Xiangya Hospital, and a signed informed consent was taken from all the patients before the study. The approval number from the human ethical committee was XCH/HEC/2015/GC14. The microbiology department of The Second Xiangya Hospital provided all the selected cell lines, which included AGS, MKN-45, BGC-823, MGC-803, SGC-7901 along with GES-1 as the normal gastric epithelial cell line. The use of the cell lines was approved by the institutional review board of The Second Xiangya Hospital. The selected cells were seeded in Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich Co., St Louis, MO, USA) which contained 1% penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Sigma-Aldrich Co.). All the cells were maintained in 5% CO₂ at 37°C.

PMNs were isolated from peripheral blood donated by healthy volunteers. The collected peripheral blood was overlaid on lymphocyte separation medium (LSM), followed by centrifugation of the layered solution at 800 × g at 21 °C for 30 min. The bottom red layer was resuspended in 3% Dextran-PBS solution for 30 min. Afterwards, the supernatant was transferred into a fresh tube and centrifuged to lyse the red blood cells and enrich the PMNs. The enriched PMNs were further sorted using the flow cytometer (Galios; Beckman Coulter, Roissy, France). The PMNs with a purity > 95% were cultured in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich Chemical Company, St Louis, MO, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) [34] (link).
The PMNs were seeded into a 24-well plate. When cells were 50% - 60% confluent, the transfection was conducted in accordance with Lipofectamine 2000 protocols (Invitrogen Inc., Carlsbad, CA, USA) for 24 h. miR-21 mimic, miR-21 mimic negative control (NC), miR-21 inhibitor, miR-21 inhibitor NC, scramble sh-NC, shRNA against IL-12A or XIST (sh-IL-12A or sh-XIST), and plasmids overexpressing IL-12A or XIST were all purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, Guangdong, China).

Stock solution of ON in dimethyl sulfoxide (DMSO) (Merck Millipore, MA, USA) in concentration of 12.8 mg/mL was prepared. Stock solutions of TGF in DMSO can also be prepared with the same last concentration of ON. 50 µL from each solutions were completed to 20 mL with Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich Co, MO, USA) and 400-fold serial dilutions were applied to final concentration of 32 µg/mL. Roswell Park Memorial Institute medium, which is commonly referred to as RPMI medium or RPMI 1640, is a form of medium used in cell culture and tissue culture used to grow a variety of mammalian cell lines. RPMI 1640 uses a bicarbonate buffering system and differs from most of the mammalian cell culture media in its typical pH 8 formulation.

5-FU was provided by Shandong Qilu Pharmaceutical Co., Ltd. (Jinan, People’s Republic of China). CDDP was provided by Nanjing Pharmaceutical Factory Co., Ltd. (Nanjing, People’s Republic of China). Glyceryl monostearate was purchased from Shanghai Chineway Pharmaceutical Tech. Co., Ltd. (Shanghai, People’s Republic of China). Injectable soybean oil was purchased from Guangzhou Hanfang Pharmaceutical Co., Ltd. (Guangzhou, People’s Republic of China). Injectable soya lecithin was obtained from Shanghai Taiwei Pharmaceutical Co., Ltd (Shanghai, People’s Republic of China). HA was provided by Shandong Freda Biochem Co., Ltd. Stearic acid, Tween^® 80, dimethyldioctadecylammonium bromide, and Roswell Park Memorial Institute 1640 medium were purchased from Sigma-Aldrich Co., Ltd (St Louis, MO, USA). All other chemicals were of analytical or higher grade.