T25 flask

Manufactured by Thermo Fisher Scientific

Sourced in United States, Denmark, Japan, United Kingdom, Germany

The T25 flasks are a type of cell culture vessel used for the in vitro cultivation of cells. They provide a surface area of 25 cm² for cell attachment and growth. The flasks are made of transparent, tissue culture-treated polystyrene and feature a vented cap to allow gas exchange.

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86 protocols using t25 flask

Culturing HNSCC and NK92MI Cell Lines

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All HNSCC cell lines were cultured and expanded in T‐25 flasks (ThermoFisher) using RPMI 1640 medium enriched with 10% Fetal Bovine Serum (HyClone, USA) and 1% Penicillin Streptomycin (ThermoFisher). Cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Medium was changed every 2–3 days and the cells were passaged when reaching about 80% of confluency (~every 3 days). NK92MI cells (ATCC, USA) were cultured and expanded in T‐25 flasks (ThermoFisher) using alpha‐Minimum Essentials Eagle Medium (MEM) enriched with 12.5% FBS (HyClone, USA), 12% horse serum (ThermoFisher), 4% MEM vitamin solution (ThermoFisher) and 0.1 mM beta‐mercaptoethanol (Thermofisher).

Tostado C.P., Da Ong L.X., Heng J.J., Miccolis C., Chia S., Seow J.J., Toh Y, & DasGupta R. (2024). An AI‐assisted integrated, scalable, single‐cell phenomic‐transcriptomic platform to elucidate intratumor heterogeneity against immune response. Bioengineering & Translational Medicine, 9(2), e10628.

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Isolation and Culture of Primary Müller Cells

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7 pellets were collected by spinning at 210g for 10 minutes at 37°C and then dissociated in fresh GM by triturating with plastic serological pipettes. Cell suspension was cultured in a T25 flask (Thermo Fisher Scientific) in a humidified incubator with 5% CO 2 at 37°C. The GM was left unchanged for the initial 4 days; GM was replenished on day 5. After 7-8 days, cellular aggregations, which are attached to the base of the flask, were removed by agitating the media and the GM was replenished. Until cells reached 90% confluency, cells were detached with 0.25% trypsin/EDTA from a T25 flask and then expended in a T75 flask (Thermo Fisher Scientific). After reaching 90% confluency in a T75 flask, cells were subcultured in the appropriate plates for subsequent experiments. Primary Müller cell characteristics were confirmed using immunolabelling for vimentin, S100β, glutamate synthetase (GS) (data not shown).

Lu Y.Z., Natoli R., Madigan M., Fernando N., Saxena K., Aggio-Bruce R., Jiao H., Provis J, & Valter K. (2017). Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration. Experimental eye research, 165.

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Differentiation of THP-1 Cells into Macrophages

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THP-1 cells (Human acute monocytic leukemia cell line) purchased from American Type Culture Collection, TIB-202, were cultured in T25 flasks (Fisher Scientific) with RPMI 1640 culture medium supplemented with 10% Fetal Bovine Serum (FBS) (Omega Scientific), β-mercaptoethanol, 100 μg/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. In recent years, these cells have been widely established as an in vitro model for native monocyte-derived macrophages in studies of inflammatory disease by particle inhalation [48 ,49 (link)]. Prior to the experiments, the monocytes were differentiated into adherent macrophages by treatment with 50 ng/ml phorbol 12-myristate-13-acetate (PMA) for 3 days.

Premshekharan G., Zhang H., Nguyen K., Forman H.J, & Leppert V.J. (2017). Low Dose Inflammatory Potential of Silica Particles in Human-Derived THP-1 Macrophage Cell Culture Studies – Mechanism and Effects of Particle Size and Iron. Chemico-biological interactions, 272, 160-171.

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Murine iPSCs Culture and Differentiation

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Murine iPSCs were routinely cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in T25 flasks (Fisher). Culture medium consisted of high glucose Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 1% non-essential amino acid, 1% Anti-Anti, 15% fetal bovine serum (FBS), and 0.1 mM β-mercaptoethanol (all Invitrogen). In order to maintain iPSC pluripotency, culture medium was supplemented with 1000 U/ml leukemia inhibitory factor (LIF). Cells were routinely passaged upon reaching approximately 80% confluence every third to fourth day and maintained in a humidified incubator with 5% CO₂ at 37 °C. One passage before the cells were added to the collagen scaffold, they were cultured on gelatine (0.1%, Fisher) coated flasks to remove excess MEFs.

Ferrie L., Premnath P., Olsen A., Larijani L., Besler B.A., Rancourt D.E., Duncan N.A., Underhill T.M, & Krawetz R.J. (2023). Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury. Scientific Reports, 13, 9378.

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3D Breast Carcinoma Cell Culture

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GFP-expressing MDA-MB-231 breast carcinoma cells were cultured in DMEM. DMEM was supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. Cells were grown in T25 flasks (Thermo Fisher Scientific) until 90% confluent. Then they were removed from the flask surface by incubation with trypsin/EDTA (Gibco) for 2 min, centrifuged, and then resuspended in a 2.5 mg/mL collagen I solution, before seeding into the 3D microfluidic devices.

Kim M.C., Li R., Abeyaratne R., Kamm R.D, & Asada H.H. (2022). A computational modeling of invadopodia protrusion into an extracellular matrix fiber network. Scientific Reports, 12, 1231.

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Xenoplantation of HS-MM cells and NK cell therapy

Cited 2 times

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Xenoplantation of HS-MM cells was performed as previously described (9 (link),10 (link)). Briefly, SCID-Beige mice were injected with 2.5x10⁷ HS-MM cells into the aponeuroses of the thighs, subsequently 9 mice were randomly divided to three groups, of three mice. A total of 0.3 ml saline, with or without 1x10⁴ murine or 1.5x10⁶ human NK cells was intravenously injected to SCID-Beige mice two weeks following HS-MM xenoplantation. A total of 8 weeks following xenoplantation, the mice were sacrificed, and the tumor size was measured using a caliper and the following formula: Volume=(width² x length)/2.
NK cells were harvested from splenic cells of BALB/c mice using a mouse NK cell isolation kit (Stemcell Technologies, Inc.), or collected from peripheral blood samples from a healthy volunteer using negative selection and a NK Cell Isolation kit, MACS system (Miltenyi Biotec, Inc.) according to the manufacturer's protocol. Written informed consent was provided from the healthy volunteer prior to the start of the study. The study was approved by the Committee of Gifu Graduate School of Gifu, Japan (approval no. 27-80, 2020-066).
The isolated human NK cells were cultured in T25 flasks (Thermo Fisher Scientific, Inc.) with 10 ml KBM502 medium (Kohjin-bio, Co., Ltd.) at 37˚C in a humidified incubator with 5% CO₂ for seven days.

Hanamatsu Y., Saigo C., Kito Y, & Takeuchi T. (2020). An obstructive role of NK cells on metastatic growth of clear-cell sarcoma cells in a xenoplant murine model. Molecular and Clinical Oncology, 14(1), 9.

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Adenomyosis-Derived ESC Isolation and Manipulation

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ESCs were extracted from myometrium of patients with adenomyosis. Components of culture medium (50 mL): Dulbecco’s Modified Eagle Medium (DMEM)/F12K (Gibco) + 10% fetal bovine serum (FBS, Gibco), 2 mmol glutamine + 1% penicillin/streptomycin solution (100X, Solarbio). Immunohistochemistry was employed to detect cytokeratin and vimentin, and interstitial cells detected were cultured. Cells were inoculated into T25 flasks (Thermo Fisher). Preheated medium (5 mL, 37°C) was added to the flasks. The cells were cultured in a 37°C and 5%CO₂ incubator (Binder) to their good growth. Before transfection, the medium was replaced with FBS-free medium. During transfection, 1×10⁵ cells per well were inoculated into 6-well plates. LncRNA H19-ad (over-expression vector), miR-17 inhibitor/mimics, TLR4 siRNA, NC vector were all purchased from Shanghai Sangon Bioengineering Co., Ltd. Cell lines were transfected using the Lipofectamine 2000 transfection kit (invitrogen, USA). After transfection for 8 h, fresh culture medium was used to remove dead cells.

Liang N., Zhang W., Wang H., Shi W., Wang L, & Ma L. (2020). Levonorgestrel Ameliorates Adenomyosis via lncRNA H19/miR-17/TLR4 Pathway. Drug Design, Development and Therapy, 14, 3449-3460.

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Isolation and Culture of Human Nasal Epithelial Cells

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Our study was performed at the Central Adelaide local health network human research ethics committee (CALHN HREC) (HREC/18/CALHN/69), with ethics approval and written consent obtained from patients prior to the collection of primary human nasal epithelial cells (HNECs). HNECs were obtained from the inferior turbinate surface with sterile nasal brushes from CRS patients who were undergoing endoscopic skull base surgery. Nasal brushings were suspended in nasal epithelial growth media (STEMCELL Technologies Australia Pty. Ltd, Tullamarine, VIC, Australia). Extracted cells were then depleted of monocytes using anti-CD68 (Dako, Glostrup, Denmark) coated culture dishes. HNECs were expanded in T-25 flasks (ThermoFisher Scientific, Waltham, MA, USA) in routine cell culture conditions of 37^∘C humidified air with 5% CO₂ in collagen-coated flasks (ThermoFisher Scientific) as a monolayer culture.

Ramezanpour M., Bolt H., Hon K., Shaghayegh G., Rastin H., Fenix K.A., Psaltis Alkis J., Wormald P.J, & Vreugde S. (2022). Characterization of human nasal organoids from chronic rhinosinusitis patients. Biology Open, 11(8), bio059267.

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Assessing CCL-13 Cell Apoptosis and Cycle

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CCL-13 cells were cultured in T-25 flasks (160430, Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a density of 1 × 10⁵ cells/flask. Flasks were filled with 80 mL of culture medium, and the filter-cap was slowly screwed to avoid the formation of bubbles. CCL-13 cells were subjected to SMG for 72 h. Flow cytometry analysis was performed using the FITC Annexin V Apoptosis Detection Kit I (556547, BD Biosciences, San Jose, CA, USA) in BD Accuri C6 Plus (BD Biosciences, San Jose, CA, USA). To analyze cell cycle progression, CCL-13 cells were fixed with 4% paraformaldehyde (09154-85, Nacalai Tesque, Kyoto, Japan) for 15 min. CCL-13 cells were washed twice with cold PBS and resuspended in 1X Binding Buffer at a concentration of 1 × 10⁶ cells/mL. CCL-13 cells were stained with 5 µL PI (51-66211E, BD Biosciences, San Jose, CA, USA). Cell cycle analysis was performed by measuring the cellular DNA content using a flow cytometer.

Ho C.N., Tran M.T., Doan C.C., Hoang S.N., Tran D.H, & Le L.T. (2021). Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins. International Journal of Molecular Sciences, 22(9), 4550.

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Clonogenic Assay for Cell Survival

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Cell survival was assessed using clonogenic assay according to the preplating and postplating protocols (23 (link), 24 (link)). For the preplating protocol, cells were resuspended in the growth medium containing Foscan (0.06 µM) and aliquots were seeded in 6-well plates (Thermo-Fisher Scientific) in triplicate in sufficient numbers to give rise to approximately 50 colonies per well. After overnight incubation, cells were irradiated. Other treatments and inhibitors were added prior to irradiation, as described above. For the postplating protocol, cells were grown in T25 flasks (Thermo-Fisher Scientific) and treated with FoscanPDT (0.15 µM Foscan + 400 mJ/cm²)±LCL29 or HPR. Overnight incubation with Foscan was followed by addition of other treatments and irradiation. After treatments, cells were trypsinized and aliquots were plated in triplicate in sufficient numbers to give rise to approximately 50 colonies per well. For both protocols, after 10 days of incubation, colonies were stained with crystal violet (0.1%) and counted. Plating efficiency was 28% (n=42).

Boppana N.B., Kraveka J.M., Rahmaniyan M., Li L., Bielawska A., Bielawski J., Pierce J.S., Delor J.S., Zhang K., Korbelik M, & Separovic D. (2017). Fumonisin B1 Inhibits Endoplasmic Reticulum Stress Associated-apoptosis After FoscanPDT Combined with C6-Pyridinium Ceramide or Fenretinide. Anticancer research, 37(2), 455-463.

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