Plastic tissue culture flasks

Manufactured by Corning

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Plastic tissue culture flasks are laboratory equipment used for the in vitro cultivation and propagation of cells, tissues, or organisms. They provide a controlled environment for cell growth and experimentation.

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9 protocols using plastic tissue culture flasks

Culturing Eca-109 Human Esophageal Cancer Cells

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Eca-109 human esophageal cancer cells were obtained from Shandong Academy of Medical Sciences (Shandong, China). The cells were incubated at 37°C in 5% CO₂ in plastic tissue culture flasks (Corning Inc., Acton, MA, USA) with complete Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Gibco-BRL, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin-streptomycin (HyClone; Thermo Fisher Scientific, Rockford, IL, USA). Cobble-shaped cells began to expand after two days. At ~80% confluence, the cells were separated by digestion with 0.25% trypsin-EDTA (Gibco-BRL) and passaged into three plastic tissue culture flasks in the growth medium for expansion.

LIANG N., SONG X., XIE J., XU D., LIU F., YU X., TIAN Y., LIU Z., QIAO L, & ZHANG J. (2014). Effect of galectin-3 on the behavior of Eca-109 human esophageal cancer cells. Molecular Medicine Reports, 11(2), 896-902.

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Isolation of Neonatal Mouse Cardiomyocytes

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Neonatal mouse cardiomyocytes (NMVCMs) were isolated from the hearts of neonates of CD-1 wild-type mice (Charles River Labs). In brief, hearts were surgically removed from 1-day-old pups and digested in Hank’s Balanced Salt Solution (Gibco) with 0.046% Trypsin (Affymetrix) at 4 °C overnight. Blood cells were removed from hearts by type II Collagenase (Worthington) after shaking at 37 °C for two minutes. A heterogeneous cell population containing cardiomyocytes and fibroblasts was isolated after further digestion using type II Collagenase (Worthington) at 37 °C for seven minutes. Fibroblasts were removed by pre-plating for 1.5 hours on 75 cm² plastic tissue culture flasks (Corning) in a humidified incubator at 37 °C with 5% CO₂. Isolated cardiomyocytes were resuspended in dark medium formulated by 75% Dulbecco’s Modified Eagle Medium (DMEM) and 25% M199 medium containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10% horse serum (HyClone), 5% fetal bovine serum (Gibco), and 1% 100x Penicillin/Streptomycin/L-Glutamine solution. Media was replaced daily and on day 3, onward, a 10 μM mM solution of arabinosylcytosine (Ara-C), an antiproliferative drug to prevent noncardiomyocyte overgrowth.

Liu J., Miller K., Ma X., Dewan S., Lawrence N., Whang G., Chung P., McCulloch A.D, & Chen S. (2020). Direct 3D Bioprinting of Cardiac Micro-tissues Mimicking Native Myocardium. Biomaterials, 256, 120204.

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Cell Culture Conditions for HLEF-104 and H9c2 Lines

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Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, vol/vol), glutamine (0.15%), HEPES (10 mM, pH 7.2) and gentamicin (50 mg/ml).
Cells were grown in plastic tissue culture flasks (Corning Incorporated, Corning, NY, USA) in an atmosphere of 5% CO₂ at 37 °C and 90% humidity. Cells were reseeded twice a week. For all experiments, cells from exponentially growing cultures were used. After the cells in the monolayer reached a density of 90%, they were treated with 0.25% trypsin and EDTA and centrifuged at 3,000 g for 5 min. The supernatant was discarded, and the cell pellet was re-suspended in growth medium and the cells were plated in a 96-well plate. All experiments were performed using an HLEF-104 culture <20 passages and an H9c2 culture <25 passages.

Akentieva N.P., Sanina N.A., Gizatullin A.R., Shkondina N.I., Prikhodchenko T.R., Shram S.I., Zhelev N, & Aldoshin S.M. (2019). Cytoprotective Effects of Dinitrosyl Iron Complexes on Viability of Human Fibroblasts and Cardiomyocytes. Frontiers in Pharmacology, 10, 1277.

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Exosome uptake in IPEC-J2 cells

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Porcine small intestinal epithelial (IPEC-J2) cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified eagle medium/Ham’s F-12 in a 1:1 ratio (Invitrogen, Life Technologies, Carlsbad, CA, USA) supplemented with 5% fetal calf serum (FCS; Invitrogen) and 5 ng/mL epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA). IPEC-J2 cells were seeded at 0.5 × 10⁵ cells/mL in a 10 mL volume in plastic tissue culture flasks (75 cm² Corning, Corning, NY, USA). After reaching confluency (four days)^{79 (link)}, the cells were seeded into 6-well tissue culture plates (9.6 cm²/well) at 2.0–2.3 × 10⁵ cells/well in a 2 mL volume. The cells were allowed to adhere for 24 h, and the media were replaced every other day. When the cells were 90% confluent, we added 0.5 mL exosomes or exosome-free supernatants to each well, the equal volume PBS added as control. We determined that a 25% media substitution was optimum. Exosomes suspended in PBS and supernatants were passed through 0.45-μm and 0.22-μm membrane filters prior to incubation. IPEC-J2 cells were harvested after 8 h. we give all the in vitro experiment for three repeats test.

Lin D., Chen T., Xie M., Li M., Zeng B., Sun R., Zhu Y., Ye D., Wu J., Sun J., Xi Q., Jiang Q, & Zhang Y. (2020). Oral Administration of Bovine and Porcine Milk Exosome Alter miRNAs Profiles in Piglet Serum. Scientific Reports, 10, 6983.

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Gastric Cancer Cell Lines Cultivation

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Human gastric cancer cell lines MGC-803 and MKN-45 were obtained from Shanghai Academy of Medical Sciences (Shanghai, China). The cells were incubated at 37°C in 5% CO₂ in plastic tissue culture flasks (Corning Inc., Acton, MA, USA) with complete Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL). ANXA5, PCNA, Bcl-2, Bax, MMP-9, E-cadherin, β-actin, GAPDH, MEK1/2, pMEK1/2, ERK1/2, and pERK1/2 monoclonal antibody were purchased from Sigma (St. Louis, MO, USA) and the ANXA5 protein was bought from novoprotein (Shanghai, China); the Trizol reagent, protein assay kit, the siRNA targeted to ANXA5 and the negative control siRNA were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA); the gel image analysis system and electrophoresis apparatus were purchased from Bio-Rad (Hercules, CA, USA). The digital image analysis system and the fluorescence microscope were from Nikon (Tokyo, Japan), and the ultraviolet analyzer was from Beckman Coulter (Miami, FL, USA).

Wang X., Dai Y., Zhao Y., Li M., Zhang J., Ci Y., Wang H, & Li X. (2021). AnnexinA5 Might Suppress the Phenotype of Human Gastric Cancer Cells via ERK Pathway. Frontiers in Oncology, 11, 665105.

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Cell Culture Protocols for Vero E6, Caco-2, IPEC-J2, and HEK 293T

Cited 2 times

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Vero E6 cells (ATCC CRL-1586) were kindly provided by the Veterinary Medicine Research Center of the Da Bei Nong Group [82 (link)]. Caco-2 and IPEC-J2 (Guangzhou Jennio Biotech Co, Ltd., China). HEK 293T cells were purchased from ATCC (United States) [83 (link)]. Caco-2, and IPEC-J2, Vero and HEK 293T cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM from Life Technologies). All cells were supplemented with 10% fetal bovine serum (FBS, GIBCO), 16 mM HEPES (Life Technologies), and 100 μg/ml penicillin/streptomycin (Invitrogen) in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were routinely seeded at a density of 2 × 10⁵/mL in 25 cm² plastic tissue culture flasks (Corning) and passaged every 3–4 days for a maximum of 30 passages and regularly tested for mycoplasma contamination.

Zhang S., Cao Y, & Yang Q. (2020). Transferrin receptor 1 levels at the cell surface influence the susceptibility of newborn piglets to PEDV infection. PLoS Pathogens, 16(7), e1008682.

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Culturing HepG2 and HepB5 Cells

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HepG2 and HepB5 cells purchased from the European Collection of Cell Cultures were cultured in Minimum Essential Medium with 10% fetal bovine serum at 37°C in a humidified 5% CO₂ atmosphere using 25 cm³ plastic tissue culture flasks (Corning Incorporated, Corning, NY, USA). Cells were in the logarithmic growth phase by routine passage every 2–3 days and split when reaching confluence. No ethics statement was required from the institutional review board for the use of these cell lines.

Mocan L., Matea C., Tabaran F.A., Mosteanu O., Pop T., Mocan T, & Iancu C. (2015). Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus–ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor. International Journal of Nanomedicine, 10, 5435-5445.

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Gastric Cancer Cell Lines Cultivation

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Wang X., Dai Y., Zhao Y., Li M., Zhang J., Ci Y., Wang H, & Li X. (2021). AnnexinA5 Might Suppress the Phenotype of Human Gastric Cancer Cells via ERK Pathway. Frontiers in Oncology, 11, 665105.

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Culturing H9C2 Cardiac Cells

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H9C2 cells, derived from embryonic rat hearts, were purchased from the American Type Culture Collection. The cells were seeded in plastic tissue culture flasks (Corning, China) at a density of 1 × 10⁵/mL and cultured in Dulbecco's Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Biological Industries, BI), 100 U penicillin, and 100 μg/mL streptomycin (Gibco). Thereafter, the cells were incubated in a 5% CO₂ humidified atmosphere (Thermo, USA) at 37°C. When the cells formed a complete monolayer, they were passaged within 30 passages for the subsequent experiments.

Yin B., Lian R., Li Z., Liu Y., Yang S., Huang Z., Zhao Z., Li Y., Sun C., Lin S., Wan R, & Li G. (2021). Tea Polyphenols Enhanced the Antioxidant Capacity and Induced Hsps to Relieve Heat Stress Injury. Oxidative Medicine and Cellular Longevity, 2021, 9615429.

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