T25 flask

Manufactured by Corning

Sourced in United States, United Kingdom, Germany

The T25 flasks are a type of cell culture vessel commonly used in laboratories. These flasks have a growth area of approximately 25 cm2 and are designed to provide a suitable environment for the cultivation of cells.

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132 protocols using t25 flask

Conditioned Media Comparison for Stem Cell Cultures

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The mES cells, 4T1 cells, or SNL cells (purchased from ATCC) were cultured to 40 % confluence with each usual culture medium respectively, and the medium for ES cells and SNL cells was changed to 7 ml DMEM and 5 % FBS per T25 flask (Corning). The medium for 4T1 cells was changed to 7 ml RPMI 1640 and 5 % FBS per T25 flask (Corning). Forty-eight hours later, the supernatants were harvested and stored at –80 °C. As already described, ES cells can maintain the self-renewal and pluripotency properties in vitro, but need fibroblast feeder cells for support. In our study, SNL cells – an immortalized fibroblast cell line – were used as feeder layers to support the growth and pluripotency of mES cell in-vitro cultures. To exclude the effect made by SNL cells when the ES conditioned medium (ES-SNL-CM) was collected from the ES cells cultured on SNL cells, we used the SNL conditioned medium (SNL-CM) as a parallel control. If ES conditioned medium (ES-CM) was collected from ES cells cultured on gelatin without feeder cells, DMEM medium was used as its control. 4T1 cell conditioned medium (4T1-CM) severed as the mock control group.

He N., Feng G., Li Y., Xu Y., Xie X., Wang H., Wang Y., Ou L., Pei X., Liu N, & Li Z. (2016). Embryonic stem cell preconditioned microenvironment suppresses tumorigenic properties in breast cancer. Stem Cell Research & Therapy, 7, 95.

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Isolation of ZIKV from tissue samples

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We used monolayers of Vero cells (ATCC, Manassas, VA) to isolate infectious virus from selected tissue samples collected from the ZIKV-inoculated animals at necropsy. Briefly, up to 10⁷,tissue mononuclear cells isolated from tissues were added to a confluent monolayer of Vero cells in 6-well plates (Costar Inc., Cambridge, MA) for tissues yielding <10⁶ cells), or T25 flasks (Costar Inc.) for tissues yielding > 10⁶ cells. The co-cultures were incubated at 37°C and culture supernatants were harvested at 2, 4 and 7 days after initiation. The supernatants were assayed for the presence of ZIKV RNA by qRT-PCR (described below). A sample was considered to be positive for infectious virus if the vRNA levels steadily increased in supernatants of the corresponding co-culture. No effort was made to titer the levels of infectious virus in samples.

Carroll T., Lo M., Lanteri M., Dutra J., Zarbock K., Silveira P., Rourke T., Ma Z.M., Fritts L., O’Connor S., Busch M, & Miller C.J. (2017). Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques. PLoS Pathogens, 13(7), e1006537.

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Cytotoxicity Evaluation of Sealing Materials

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L929 mouse fibroblasts (American Type Culture Collection, Manassas, VA) were used to determine the cytotoxicity of the extracted materials. The cells were grown as monolayer cultures in T-25 flasks (Costar, Cambridge, MA) and subcultured at 37°C in an atmosphere of 5% CO₂ in air and 100% relative humidity. The culture medium was DMEM/F12 nutrient mixture (1:1) (MilliporeSigma) supplemented with 10% (v/v) fetal bovine serum and 1% gentamycin. Adherent cells at a logarithmic phase were detached with a mixture of 0.05% trypsin (MilliporeSigma) and 0.02% EDTA (MilliporeSigma) incubated for 5–10 minutes at 37°C and used for plating into a 96-well tissue culture plate (2 × 10⁴ cells in 0.1 mL growth medium/well) in triplicate. The next day, cells (L929) were incubated with the different sealing materials for 24, 48, 72, and 96 h. Following incubation, cell viability was assessed using the MTS assay as recommended by the supplier (Promega, Madison, WI). Briefly, cells were incubated with the reagents from the kit for 1 h and the absorbance in each well was measured at 490 nm. Cell viability was determined as a percentage of control.

Saghiri M.A., Karamifar K., Nath D., Gutmann J.L, & Sheibani N. (2020). A Novel Polyurethane Expandable Root Canal Sealer. Journal of endodontics, 47(4), 612-620.

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Isolation and Culture of hBMSCs

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The hBMSCs were isolated by subjecting to Ficoll (1.077 g/ml; Tianjin Haoyang Biological Products Technology Co., Ltd.) density gradient separation. The mononuclear fraction was collected and plated in T-25 flasks (Corning, Inc.) using complete DMEM/F-12 containing 10% fetal bovine serum and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) medium at 37˚C with 5% CO². After three days, the medium was changed to remove non-adherent cells. Then, the media was changed every three days. When the primary cells reached 70-80% confluence, the cells were passaged by trypsinization (Hyclone; Cyvita), and cultured based on the aforementioned method.

Song D., Wu Z.S., Xu Q., Wang K., Xu M.T., Ha C.Z., Zhang C, & Wang D.W. (2021). LRRC17 regulates the bone metabolism of human bone marrow mesenchymal stem cells from patients with idiopathic necrosis of femoral head through Wnt signaling pathways: A preliminary report. Experimental and Therapeutic Medicine, 22(1), 666.

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Generation of Medullospheres from Brain Tumor Cells

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In order to produce medullospheres (MBS) from DAOY, UW228 and ONS-76, cells were grown at confluence in adherent conditions, trypsinized, pelleted and plated (6 × 10⁴/ml) in ultra-low attachment T25 Flasks (Corning Inc., NY, USA) for a further 7 days in serum-free EUROMED CSC (EUROCLONE Spa, Pero, MI, Italy) medium. Culture conditioning based on low attachment flasks, specialized medium and formation of spheres enabled the expansion of the CSC population. Suspensions of spheres from all cell lines were collected and counted in 96-well plates by inverted microscopy (Olympus CKX41).

Bisaro B., Mandili G., Poli A., Piolatto A., Papa V., Novelli F., Cenacchi G., Forni M, & Zanini C. (2015). Proteomic analysis of extracellular vesicles from medullospheres reveals a role for iron in the cancer progression of medulloblastoma. Molecular and cellular therapies, 3, 8.

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Hypoxia Effect on SK-N-AS Cells

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SK-N-AS cells (1 × 10⁶) were incubated in either 21 or 1% O₂ for 3 days in T25 flasks (Corning, U.K.). Cells were harvested with a 2 ml PBS wash, trypsinisation and suspension in 5 ml medium. A cell count was performed at harvesting time, and approximately 5–6 × 10⁶ cells were used for each condition. Supplementary Figure S1C shows a typical confluency at harvesting time for both normoxic and hypoxic conditions. In order to remove the residual culture medium, cells were gently washed in PBS by repeated cycles of centrifugation and supernatant discard. Upon centrifugation (1200×g for 5 min), the supernatant was discarded and the pellet of cells was resuspended in 1 ml PBS, centrifuged (1200×g for 5 min) and the pellet of cells was frozen immediately in liquid nitrogen and stored at −80°C until acetonitrile:water extraction.

Al-Mutawa Y.K., Herrmann A., Corbishley C., Losty P.D., Phelan M, & Sée V. (2018). Effects of hypoxic preconditioning on neuroblastoma tumour oxygenation and metabolic signature in a chick embryo model. Bioscience Reports, 38(4), BSR20180185.

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Establishment of Melanoma Cell Lines

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To generate primary melanoma cell lines, AP2ε^-/-/Tg(GRM1) and age-matched Tg(GRM1) were sacrificed by cervical dislocation and lungs were dissected immediately. The tissue was briefly washed with 1x PBS. Subsequently, the lung tissue was cut into small pieces and was added to a mixture of 1 ml Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, München, Germany) and collagenase VIII from Clostridium histolyticum (Sigma-Aldrich, München, Germany). After incubating at 37 °C for 3 h the cell suspension was seeded into T25 flasks (Corning Inc., Corning, NY, USA). The cells were cultured in high-glucose DMEM supplemented with 10% FCS, 1% penicillin/streptomycin and 0.5 µg/mL amphotericin B (Sigma Aldrich, St. Louis, MO, USA) at 37 °C in humidified 8% CO₂. To confirm that the isolated cells are from melanocytic origin, GRM1 expression was verified by RT-PCR, as previously described [23 (link)] (Supplementary Figure S1). Human melanoma cell lines Mel Juso, 501Mel, and Mel Im (FUCCI) were cultivated as indicated elsewhere [24 (link), 25 (link)]. Mycoplasma contamination was regularly excluded for all primary cells and cell lines. Cell lines were authenticated using the short tandem repeat (STR) method.

Staebler S., Rottensteiner-Brandl U., El Ahmad Z., Kappelmann-Fenzl M., Arkudas A., Kengelbach-Weigand A., Bosserhoff A.K, & Schmidt S.K. (2024). Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma. Cell Death & Disease, 15(5), 351.

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Culturing and Passaging Human Embryonic Stem Cells

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hESCs were cultured and passaged as our previous paper described [11 (link)]. In brief, hESCs were cultured with the Irradiated CF1 feeder cells (3×10⁴ cells/cm²) on the T25 flasks (Corning) coated with Matrigel (Becton–Dickinson). hESCs were maintained in DMEM/F12 (Invitrogen) supplemented with 20 % knockout serum replacement (Invitrogen), 4 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), 2 mmol/L l-glutamine (Invitrogen), 1 % nonessential amino acids (Invitrogen) and 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich). hESCs were passaged approximately once a week. Collagenase IV was used to dissociate the cells from the feeders as cell clumps, which were dissociated to an appropriate size before being passaged onto newly prepared feeder cells.

Chen H., Li Y., Lin X., Cui D., Cui C., Li H, & Xiao L. (2015). Functional disruption of human leukocyte antigen II in human embryonic stem cell. Biological Research, 48, 59.

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Cell Culture Expansion in Bioreactors

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T-25 flasks (Corning) were seeded with approximately 2.5 x10⁴ cells/cm² in a prewarmed MEM supplemented with 10% fetal bovine serum (FBS). The seeded flasks were incubated in an incubator at 37°C with 5% CO₂ condition. Daily observation of each flask was conducted at least twice using an inverted microscope (Olympus® Japan) for complete monolayer formation. Following confluence, the cells were washed with prewarmed Ca²⁺/Mg²⁺ free Dulbecco's PBS (D1408 SIGMA) and detached from the flask with StemPro®Accutase® (A11105-01 Gibco, USA) whose dissociation activity was not stopped until all the cells became detached from the flasks. The dissociated cells were then used to seed the bioreactors.

Lawal N., Hair-Bejo M., Arshad S.S., Omar A.R, & Ideris A. (2018). Propagation and Molecular Characterization of Bioreactor Adapted Very Virulent Infectious Bursal Disease Virus Isolates of Malaysia. Journal of Pathogens, 2018, 1068758.

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Cell Culture and Maintenance in DMEM

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All cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco, Life Technologies, Paisley, UK) with added 10% foetal bovine serum (FBS) (GE Healthcare, UK), 2 mM glutamine (Sigma-Aldrich, Dorset, UK), 100 units/ml penicillin and 100 units/ml streptomycin (Invitrogen, Life Technologies, Paisley, UK). LS174T^pcDNA/BCL-3 and SW620^pcDNA/BCL-3 cell lines were cultured in 10% DMEM with added 400 µg/ml G418 (Sigma-Aldrich). For stock purposes, cells were maintained in T25 flasks (Corning, NY, USA) and incubated at 37°C in dry incubators maintained at 5% CO₂. Cells media were changed every 3-4 days.

Legge D.N., Shephard A.P., Collard T.J., Greenhough A., Chambers A.C., Clarkson R.W., Paraskeva C, & Williams A.C. (2019). BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells. Disease Models & Mechanisms, 12(3), dmm037697.

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