Three cell lines—A549, HeLa and HEK293T—were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBA, Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (GE Heathcare Life Sciences, Chicago, IL, USA). All cells were cultured in 5% CO2 in a 37 °C incubator (SANYO, Osaka, Japan).
37 c incubator
Manufactured by Sanyo
Sourced in Japan
The 37°C incubator is a laboratory equipment designed to maintain a constant temperature of 37°C, which is the standard temperature for many biological and medical applications. It provides a controlled environment for the growth and cultivation of cell cultures, bacteria, and other microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Merck Group (1 mentions)
Optipro serum free medium, by Thermo Fisher Scientific (1 mentions)
Mdck cells, by Thermo Fisher Scientific (1 mentions)
Mdck cells, by American Type Culture Collection (1 mentions)
Matrigel, by BD (1 mentions)
Transwell plate, by Corning (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Mtt kit, by Genview (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Antibiotic antimycotic mix, by Merck Group (1 mentions)
Streptomycin, by Beyotime (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Mitotempo, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Beas 2b, by National Collection of Authenticated Cell Cultures (1 mentions)
9 protocols using 37 c incubator
1
Culturing A549, HeLa, and HEK293T Cells
2
Influenza Virus Propagation in MDCK Cells
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Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO2: 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated).
Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56 (link)]; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57 (link)] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].
Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56 (link)]; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57 (link)] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)].
3
Assessing Cell Viability and Invasion
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Cell viability was assessed at the indicated time points using an MTT kit (Gen-View, Calimesa, USA). MTT reagent (20 μL/well) was added to the medium, and the absorbance was read using a microplate reader at 490 nm for the test length and 570 nm for the reference. The percentage of viable cells was calculated based on the absorbance of the PBS control. Inserts in 24-well Transwell plates (Corning Costar, New York, USA) were coated with 500 μL/well Matrigel (BD Bioscience, San Jose, USA). Rehydration was performed with 500 μL/well serum-free medium for 2 h in a 37°C incubator (Sanyo, Japan). Samples were seeded into the inserts after removal of the rehydration medium. After 24 h of incubation, the cells that invaded through the membrane were stained with crystal violet (Beyotime, Shanghai, China) and observed at 200× magnification using a light microscope (Olympus, Tokyo, Japan).
4
Primary Hepatocyte Isolation and Culture
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Primary hepatocytes were dissociated from anesthetized adult mice by a non-recirculating collagenase perfusion through theportal vein. The isolated cells were then filtered through a 100 μm pore size mesh nylon filter, and 5*10^5 cells grown in 3.5 cm petri-dishes cultured with hepatocyte medium (HM) (ScienCell, UT, USA), supplied with 5% fetal bovine serum (FBS), 1% Penicillin–Streptomycin (P/S), in a 37 °C incubator (Sanyo, Guangzhou, China) containing 5% carbon dioxide.
5
Determining Optimal CBX Pretreatment and Mechanical Stress Effects on MSCs
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First, a safe concentration of CBX (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was determined using the CCK8 method according to the instructions provided with the CCK-8 cell proliferation and cytotoxicity assay kit (cat. no. CA1210; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). This concentration (100 µM) was used for subsequent experiments. MSCs were divided into 6 groups: A control group that was neither stressed nor treated with CBX; a stress group, which was only treated with stress; and four groups that were stressed after CBX treatment for 3, 6, 12, and 24 h. Stress loading was performed using a four-point bending device, as previously described (15 (link)). The stress loading strength was 4,000 µ strain with a frequency of 0.5 Hz for 15 min. The treated MSCs were cultured in DMEM with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) in a 37°C incubator (SANYO, Osaka, Japan).
6
HeLa Cell Cultivation and Virus Propagation
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The human cervical epithelial carcinoma cell line (HeLa) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, Vienna, Austria) supplemented with 10% fetal bovine serum (Sigma Aldrich) and 1% antibiotic–antimycotic mix (Sigma Aldrich) in a 37°C incubator (Sanyo, Okayama Prefecture, Japan; CO2: 5%, relative humidity: >95%). In all experiments a medium containing 2% fetal bovine serum and 1% antibiotic–antimycotic mix was used. HRV1a and 8 serotypes were obtained from the ATCC and grown on HeLa cells. The stocks were frozen at −80°C and virus titers were determined by 50% tissue culture infective dose (TCID50) assay.
7
Culture and Hypoxia Conditions for A549 and H1975 Cells
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Adenocarcinoma human alveolar basal epithelial cells (A549, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) and Human lung adenocarcinoma cells (H1975, Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were used in this study. Both of them have passed the STR authentication by Guangzhou Cell cook Biotech Co., Ltd. Both cell lines were cultured in RPMI-1640 medium (HyClone, USA) containing 10% fetal bovine serum (FBS, HyClone), 100 units antibiotics penicillin per ml and 100 µg streptomycin per ml (Beyotime Biotechnology, Shanghai, China). Cells culture at 37°C incubator (Sanyo Electric Co., Ltd., Japan) with 5% CO2 which provides normoxia conditions. The hypoxia situation (pO2: 5%) was achieved in a hypoxia incubator (MCO-170MUVL, Panasonic, Japan) with a mixture of 5% O2, 5% CO2 and 90% N2. Cells were washed by phosphate buffer saline (PBS) and digested by 0.25% trypsin and 0.02% EDTA solution (Genom biomedical technology Co., Ltd., China).
8
Viral Infection and Mitochondrial Modulators
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HeLa and Vero cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Corning, NY, USA), supplemented with 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA) and heat-inactivated 10% FBS in a 37°C incubator (SANYO Electric Co, Osaka, Japan) with 5% CO2. CVB3 (VR-30, ATCC) was used to infect Vero and HeLa cells at 37°C. The CVB3 titer was determined using plaque assays. Mitoquinone (MitoQ) was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA). MitoTEMPO and N-acetyl-l -cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Cell Lines and Culture Conditions
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Human NSCLC cells (H1975, H1299 and PC9), large cell lung cancer cells (H460), lung mucosal epithelial cells (H292), normal lung epithelial cells (BEAS-2B) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Lung cancer cells were maintained in RPMI-1640 medium, and BEAS-2B cells were maintained in DMEM medium. Mediums were supplied with 10% fetal bovine serum, 100 mg/mL streptomycin and 100 units/mL penicillin. Cells were cultured in a 37 °C incubator (Sanyo, Japan) with 5% CO2. All cells passed the short tandem repeat (STR) analysis of Guangzhou Cellcook Biotech Co., Ltd, China.
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