A549 cells, an alveolar epithelial cell line (Sigma Aldrich, Dorset, UK), were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), supplemented with fetal bovine serum (FBS), L-glutamine and antibiotics (Lonza, Basel, Switzerland). Passage number was less than 20. BEAS-2B, a bronchial epithelial cell line, was a gift from Dr Nicolas Mercardo (National Heart and Lung Institute, Imperial College London, UK). BEAS-2B cells were maintained in Roswell Park Memorial Institute-1640 media containing HEPES (RPMI, Life Technologies, Paisley, UK) supplemented with FBS L-glutamine and antibiotics. Passage number was less than 20. Cells were maintained in an incubator set to 37°C and 5% CO2. The type 2 Streptococcus pneumoniae (S. pneumoniae) encapsulated strain D39 was purchased from the National Collection of Type Cultures (NCTC 7466, Central Public Health Laboratory, UK) and grown in liquid culture brain heart infusion broth (Oxoid, Basingstoke, UK). Pneumococci were grown at 37°C in a water bath to mid-logarithmic phase (optical density 600 = 0.4 to 0.6) before use in cell infection experiments.
A549 cells
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
A549 cells are a type of human alveolar basal epithelial cell line derived from a lung carcinoma. They are commonly used in research as a model for studying lung biology and disease, as well as for testing the effects of various compounds on lung cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
A549 cell, by American Type Culture Collection (4 mentions)
Rpmi 1640, by Merck Group (3 mentions)
Skov3 cells, by American Type Culture Collection (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Lonza (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
Media 199, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a medium, by American Type Culture Collection (1 mentions)
Rpmi 1640 media, by American Type Culture Collection (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by USA Scientific (1 mentions)
Hek293ft cells, by American Type Culture Collection (1 mentions)
19 protocols using a549 cells
1
Cell Culture and Pneumococcal Infection Protocol
2
Isolation and Culture of Neonatal Rat Ventricular Myocytes
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Neonatal Rat Ventricular Myocytes (NRVMs) were isolated from 1-2 day-old neonatal rat pups as described previously7 (link),19 (link),38 (link),39 and plated at a density of 200,000 cells per well of a 6-well culture dish. NRVMs were cultured in Media 199 (GIBCO) supplemented with the following: 1% HEPES Buffer solution (GIBCO), 1% MEM non-essential amino acids (GIBCO), 1.75g Glucose, 1% 200 mM L-glutamine (GIBCO), 10% or 2% heat inactivated fetal bovine serum (GE Healthcare). SKOV-3 cells (ATCC) were cultured in McCoy’s 5A medium (ATCC) supplemented with 10% heat inactivated fetal bovine serum (GE Healthcare). 769-P cells (ATCC) were cultured in RPMI-1640 media (ATCC) supplemented with 10% heat inactivated fetal bovine serum (GE Healthcare). A549 cells (Sigma-Aldrich) were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham media (Sigma-Aldrich) supplemented with 10% heat inactivated fetal bovine serum (GE Healthcare).
3
Virus Infection Protocols in Cell Lines
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Experiments were carried out in HEK 293 FT cells (ATCC), MDCK cells (ATCC), or A549 cells (Sigma). Cells were grown in Dulbecco’s Minimum Essential media (Gibco) supplemented with 10% Fetal Bovine Serum (USA Scientific) and Penicillin/Streptomycin (Gibco) (Growth media). Virus infections were carried out in Dulbecco’s Minimum Essential media (Gibco) supplemented with 0.3% Bovine Serum Albumin Fraction V (Gibco), Penicillin/Streptomycin (Gibco), and 1 µg/mL L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) treated Trypsin (Sigma) (Infection media). Minigenome experiments were carried out in Optimem (Gibco) without supplementation. Plaque purifications were performed using warmed overlays of 20% 10X DMEM (Gibco) in ddH20 supplemented with Bovine Serum Albumin Fraction V (Gibco), Glutamax™ (Gibco), gentamycin (Gibco), 5% NaCO3 (Gibco), 1 µg/mL TPCK treated Trypsin (Sigma), and Seakem™ LE (low melting point) Agarose (Lonza) (Purification media).
4
Investigating Mitochondrial ROS in A549 Cells
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A549 cells were obtained from the American Type Culture Collection (ATCC). Cells were grown in RPMI 1640 medium (Gibco) containing 10% inactivated foetal bovine serum (FBS; Gibco). Cells were seeded at a concentration of 2 × 105 in 0.5 mL of medium using sterile 6-well culture plates. The A549 cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and treated with PP (1, 2, and 4 mg/mL) for 16 h. The mitochondrial ROS inhibitor N-acetyl-L-cysteine (NAC; Sigma, A7250) was added 6 h before PP treatment. A p38 MAP kinase inhibitor (Sigma, SML0543) was added 2 h before PP treatment (Additional file 3 ).
5
Influenza A Virus Infection in A549 Cells
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A549 cells (American Type Culture Collection, ATCC) were seeded at 2.5 × 105 cells per well (0.5 mL per well) in a 24-well plate in F-12K medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C overnight. Influenza A/Brisbane/59/2007 (H1N1), originally obtained from the National Institutes of Health (NIH) Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious Diseases (NIAID), NIH (NR-12282; lot 58550257), was propagated in Madin–Darby canine kidney cells (ATCC) for three passages. Virus was added to A549 cells at a multiplicity of infection of 1 in 100 µL of influenza virus growth medium [DMEM with 100 U/mL penicillin, 100 mg/mL streptomycin, 0.2% bovine serum albumin, and 0.2 µg/mL TPCK-treated-trypsin (Sigma-Aldrich)]. After incubation at 37°C for 1 h, cells were washed once with Hank's Balanced Salt Solution and 500 µL of influenza virus growth medium was added to each well. Cells were again incubated at 37°C, and at 24 h following infection, supernatants from four wells were pooled together and cells were harvested in TRIzol reagent. Minimal cytopathic effect was observed in A549 cells postinfection. The experiment was repeated once with cells collected at 12 h postinfection instead of 24 h.
6
Cytotoxicity and Molecular Responses of A549 Cells to ZnO Nanoparticles
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Human alveolar lung epithelial A549 cells obtained from the American Type Culture Collection (ATCC, USA) were cultured in RPMI-1640 medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin and L-glutamine. For the study of cytotoxic effects, 10,000 cells in 100 μL were seeded into 96-well tissue culture plates (Sarstedt, Germany). After allowing for overnight attachment, the cells were exposed to ZnO NP concentrations (0.2 – 200 μg/mL) for 24 hr. For the study of mRNA up-regulation, release of cytokines and immunoblotting experiments, 106 cells per well seeded into 6-well tissue culture plates were used. Subsequently cells were exposed to a dose of 20 μg/mL of the ZnO NPs for 1, 2, 4, 6 or 24 hr. ZnCl2 was used as a non-engineered form of zinc particulate control.
7
A549 Cell Culture Protocol
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A549 cells (#86012804 Sigma, Taufkirchen, Germany), a human lung (carcinoma) line, were grown in Ham’s F-12K (Kaighn’s) medium (#21127022; Gibco/Thermo Fisher Scientific, Dreieich, Germany), supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 4 mM glutamine. The cells were incubated in 96-well plates/24-well plates in a humidified atmosphere of 5% CO2 in air (37 °C).
8
Cigarette Smoke and COPD Exacerbation in A549 Cells
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A549 cells, pulmonary adenocarcinoma derived cell line [18 (link)], purchased directly from Sigma- Aldrich (catalog number 86012804) and cultured in Dulbecco’s modified eagle’s medium (DMEM), (Biological Industries, Beit Haemek, Israel) with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 mg/ml streptomycin, under a humidified atmosphere (5% CO2 plus 95% air) at 37°C. Treatments included 2%, 4% and 10% CSE. LPS was added to the cultures to stimulate COPD exacerbation condition [6 (link)].
9
Cell Culture Protocol for Cancer Cell Lines
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Cells were obtained from the American Type Culture Collection (ATCC) or collaborators. Cells were passaged twice weekly to maintain a confluency at less than 70% until used in an assay. All cells were cultured at 37 °C with 5% CO2. SKOV3 cells (obtained from ATCC) were cultured in DMEM (Sigma-Aldrich, Gillingham, UK), A549 cells (also obtained from ATCC) were cultured in RPMI 1640 (Sigma-Aldrich, Gillingham, UK) and BT-20 cells (Obtained from Dr Lynda Coughlan, University of Glasgow) were cultured in α-MEM (Gibco, ThermoFisher Scientific, Waltham, USA). All media was supplemented with 10% fetal bovine serum and 2% penicillin and streptomycin. RPMI 1640 was also supplemented with 1% L-glutamine.
10
Lung Cancer Cell Culture Conditions
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Human lung cancer A549 cells (ATCC, Manassas, VA, USA) stably expressing the vector alone (A549-Vec) or CUG2 (A549-CUG2), and A549-CUG2 cells with stably silenced STAT1 (A549-CUG2-shSTAT1) or the control (A549-CUG2-shVec) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, and G418 (0.5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C with 5% CO2. For A549-CUG2-shSTAT1 and A549-CUG2-shVec cells, puromycin (1 µg/ml; Sigma-Aldrich; Merck KGaA) was additionally added to the medium.
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