The PC cell lines PANC-1 (catalog no: ATCC CRL-1469) and Capan-2 (catalog no: ATCC HTB-80) were purchased from the American Type Culture Collection (ATCC, USA). PANC-1 and Capan-2 cells were cultured at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). To create a hypoxic microenvironment, PC cells were placed in a three-gas incubator (Thermo Fisher Scientific, USA), and the oxygen concentration was set at 1%. The Affiliated Hospital of Guizhou Medical University provided 48 paired clinical PC samples, collected between January 2019 and December 2020, for this study. The Human Research Ethics Review Committee of Guizhou Medical University approved the application of these clinical samples (approved number: 2022–11), which was performed according to the tenets of the Declaration of Helsinki.
Three gas incubator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The three-gas incubator is a laboratory equipment designed to provide a controlled environment for cell culture or other applications that require precise control of temperature, humidity, and gas composition. The core function of this incubator is to maintain a stable and consistent environment within the chamber to support the growth and development of cells or other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Capan 2, by American Type Culture Collection (1 mentions)
Panc 1, by Thermo Fisher Scientific (1 mentions)
Capan 2, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Beyotime (1 mentions)
5 protocols using three gas incubator
1
Hypoxic Culture of Pancreatic Cancer Cells
2
Hypoxia and TGF-β1 Effects on Dermal Fibroblasts
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Human adult dermal fibroblasts (lot no. 61447289) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and seeded at a density of 10,000 cells/cm2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic-antimycotic in a humidified incubator at 37°C with 5% CO2. To induce hypoxia, the cells were placed in three-gas incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) that maintains a sub-ambient O2 level (1%, 5% or 10%) with or without 10 ng/ml of TGF-β1 (Peprotech, Rocky Hill, NJ, USA) by the regulated injection of N2 for 48 h. The control cells were placed in a similar incubator which was maintained at 5% CO2 and 21% oxygen level. All reagents were purchased from Invitrogen (Carlsbad, CA, USA) unless otherwise stated.
3
Hypoxia-induced Myofibroblast Transdifferentiation
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To determine if myopia risk genes could mediate hypoxia-induced myofibroblast transdifferentiation and inhibition of collagen production, HSF cultures were established as previously described [23] (link). HSFs were cultured in a 5% CO2 humidified incubator at 37°C in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 2 mM GlutaMAXTM (Gibco). The cells were seeded in 6-well culture plates, at a density of 2.5 × 105 cells/mL for 24 hours, and then transfected with small interfering RNAs (siRNAs) using Lipofectamine® RNAiMAX reagent (LipoRNAiMax, Invitrogen), according to the manufacturer's instructions. Sequences of siRNAs targeting the human genes tested (APLP2, CLUSTERIN, GAS6, SPTBN1, and STK11) are shown in Supplementary Table 3 . Cells transfected with scrambled-sequence normal control (NC) siRNA served as controls. After incubation for 36 hours, siRNA-transfected cells were exposed to 1% O2 for 12 hours in a three-gas incubator (Thermo Fisher Scientific, Waltham, MA, USA). siRNA-transfected cells incubated under normoxia (21% O2) served as controls. At the end of the designated periods, the cells were harvested, and quantities of HIF-1A, α-SMA, paxillin, and COL1A1 proteins were determined by western blotting.
4
Protective Mechanisms of KLF2 and FGF2 in HUVECs
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The School of Pharmaceutical Science (Wenzhou Medical University, Zhejiang Province, China) provided the human umbilical vein endothelial cells (HUVECs) for this study. HUVECs were cautiously cultivated in DMEM added with FBS (10%, fetal bovine serum), penicillin (100 U/mL) and kyowamycin (100 μg/mL) under 37 °C in a humidified cell incubating device with continuous 5% CO2 supply.
During the cells' logarithmic development phase, the oxygen-glucose deprivation/re-oxygenation (OGD/R) model was developed. Initial media replacement involved switching to serum-free RPMI 1640 medium from GIBICO, and cells were then put into a three-gas incubator from Thermo Scientific that contained 95% N2 and 5% CO2, lowering oxygen levels to 1%. Cells were cultured for 8 hours, and then they were cultivated for 16 hours in a typical DMEM high-glucose medium. Prior to hypoxia, all prescribed treatments were given: lentivirus KLF2 for 48 hours and FGF2 (20 ng/mL) during 24 hours.
Cell viability was measured using CCK8 (Beyotime, Shanghai, China) reagent following manufacturer's instructions. To measure cell viability, the OD450 value was normalised to the control group.
During the cells' logarithmic development phase, the oxygen-glucose deprivation/re-oxygenation (OGD/R) model was developed. Initial media replacement involved switching to serum-free RPMI 1640 medium from GIBICO, and cells were then put into a three-gas incubator from Thermo Scientific that contained 95% N2 and 5% CO2, lowering oxygen levels to 1%. Cells were cultured for 8 hours, and then they were cultivated for 16 hours in a typical DMEM high-glucose medium. Prior to hypoxia, all prescribed treatments were given: lentivirus KLF2 for 48 hours and FGF2 (20 ng/mL) during 24 hours.
Cell viability was measured using CCK8 (Beyotime, Shanghai, China) reagent following manufacturer's instructions. To measure cell viability, the OD450 value was normalised to the control group.
5
Culturing LLC and C2C12 Cells
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Lewis lung cancer (LLC) cells were acquired from the Guangzhou University of Chinese Medicine (Guangzhou, China). C2C12 myoblasts were purchased from Fuheng BioLogy (Shanghai, China). Dulbecco's modified Eagle's medium, high glucose, L-glutamine, phenol red (DMEM, Cat. #11965092), fetal bovine serum (FBS, Cat. #10270106), penicillin/streptomycin (Cat. #10378016), trypsin-EDTA, and 0.05% phenol red (Cat. #25200072) were purchased by Gibco (NY, USA). All cells were identified by short tandem repeat (STR). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells are placed in a three gas incubator (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% CO2, 37°C constant temperature, and damp environment for culture. The medium was changed every 72 hours, and the cells were routinely subcultured when they reached 90% confluence. Logarithmic growth phase LLC and C2C12 cells were used to conduct the experiments. In order to maintain the stability of HIF-1α protein in vitro, C2C12 cells were supplemented with 0.1 μM DMOG (Cat. #D3695, Sigma-Aldrich, Darmstadt, Germany) in an in vitro experiment.
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