T75 flasks

Manufactured by Merck Group

Sourced in United States

The T75 flasks are a type of cell culture vessel used in laboratories. They have a surface area of approximately 75 cm2 and are commonly used for the growth and maintenance of adherent cell lines.

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16 protocols using t75 flasks

HNSCC and HDF Cell Culture Protocol

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A HNSCC cell line (SCC-9) was purchased from American Type Culture Collection (ATCC) (CRL-1629; Manassas, VA, USA). The cells used in this study were transformed cells and, thus, review board approval was not required. HDFs were acquired from Lonza (CC-2509; Basel, Switzerland). Cell passage numbers used were between 3 and 12. HDF cells were cultured in DMEM supplemented with 10% (v/v) FBS and a 1% (v/v) antibiotic–antimycotic solution. HNSCC cells were cultured in 1:1 DMEM:F-12 supplemented with 10% (v/v) FBS, 1% (v/v) antibiotic–antimycotic solution, and 400 ng/mL of hydrocortisone. Each cell line was cultured in T75 flasks from Sigma-Aldrich and incubated at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide.

Hassan C.E, & Webster T.J. (2016). The effect of red-allotrope selenium nanoparticles on head and neck squamous cell viability and growth. International Journal of Nanomedicine, 11, 3641-3654.

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BV2 Microglia Cell Line Culture and Stimulation

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The BV2 microglia cell line CVCL_0182 was initially obtained from Dr. Markus Kipp (formerly RWTH Aachen University, Germany; now University of Rostock, Germany), who obtained the cells from Interlab Cell Line Collection (ICLC), Italy (accession number: ICLC ATL03001; http://wwwsql.iclc.it/det_list.php). The BV2 cell line was cultured in RPMI1640 (Roswell Park Memorial Institute 1640) GlutaMAX medium containing 10% FCS, 1% penicillin/streptomycin (P/S), and maintained in poly-l-ornithine (0.01%)-coated T75 flasks (Sigma Aldrich, Taufkirchen, Germany) in a humidified incubator at 5% CO₂ and 37 °C. Cells were split every 2–3 days until passage 20. For inflammatory stimulation experiments, BV2 cells were stimulated with 20 ng/mL or 100 ng/mL mouse TNF-α (product # 300-01A, Peprotech, Hamburg, Germany) as indicated. For CSN5 mimicry or inhibition experiments, cells were pre-treated with 500 nM MLN4924 and 1 or 4 μM CSN5i-3, respectively, for 2–4 h in all experiments. Final DMSO concentrations in the vehicle groups were between 0.1% and 0.4% and did not interfere with cell viability as verified in scouting experiments.

Tian Y., Milic J., Monasor L.S., Chakraborty R., Wang S., Yuan Y., Asare Y., Behrends C., Tahirovic S, & Bernhagen J. (2023). The COP9 signalosome reduces neuroinflammation and attenuates ischemic neuronal stress in organotypic brain slice culture model. Cellular and Molecular Life Sciences, 80(9), 262.

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Thawing and Maintaining Glioblastoma Cells

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Frozen U87-EGFRvIII-Luc glioblastoma cells^{45 (link)} overexpressing EGFRvIII, as well as the luciferase gene, were thawed and maintained in RPMI media containing 10% FBS and 1% penicillin/streptomycin (Gibco) in T75 flasks (Sigma).

Chapelin F., Gao S., Okada H., Weber T.G., Messer K, & Ahrens E.T. (2017). Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model. Scientific Reports, 7, 17748.

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Culturing Human Mesenchymal Stem Cells

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Human mesenchymal stem cells from the bone marrow (MSC) of a 30-year-old female donor were purchased from PromoCell (Heidelberg, Germany) and cultured in MSC growth medium 2 (MSC-GM2, PromoCell), with supplement mix (PromoCell) and 1% penicillin/streptomycin. Cells were seeded at a density of 5000/cm² in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis, MO, USA), at 37 °C in a humidified atmosphere with 5% CO₂. The culture medium was changed every two days and cells were trypsinized when almost 80% confluent.

Giordani C., Matacchione G., Giuliani A., Valli D., Scarpa E.S., Antonelli A., Sabbatinelli J., Giacchetti G., Sabatelli S., Olivieri F, & Rippo M.R. (2023). Pro-Osteogenic and Anti-Inflammatory Synergistic Effect of Orthosilicic Acid, Vitamin K2, Curcumin, Polydatin and Quercetin Combination in Young and Senescent Bone Marrow-Derived Mesenchymal Stromal Cells. International Journal of Molecular Sciences, 24(10), 8820.

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TMZ Resistance in U251 Glioma Cells

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The U251 cells were purchased from the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified incubator (Sanyo, Osaka, Japan) with 5% CO₂ at 37℃. Basal cell culture was maintained in T-75 flasks (Sigma-Aldrich, St. Louis, MO, USA). Cells were trypsinized when they reached 80-90% confluence and were seeded into 6-well plates. TMZ was added to the 6-well plates at an initial concentration of 0.25 µg/mL. The concentration was doubled after the cells were cultured for 15 days, and the final concentration of TMZ was 16 µg/mL. After withdrawal of TMZ for 2 months, the cells were re-challenged with 16 µg/mL TMZ for 1 week every month.

Guo Z., Chen Q., Liu B., Tian D., Zhang S, & Li M. (2014). LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells. Yonsei Medical Journal, 55(5), 1196-1205.

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Murine ESC-D3 Pluripotency Maintenance

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Murine ESC-D3 (ATCC, Manassas, VA) were cultured on 0.1% gelatin (Sigma, St. Louis, MO) coated T-75 flasks (Sigma, St. Louis, MO) in high glucose Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), 1% non-essential amino acid (NEAA), 10,000 U/mL of penicillin and 10,000 μg/mL streptomycin, and 0.1 mM ß-Mercaptoethanol (ßME) (all Invitrogen, Carlsbad, CA). To maintain mESC pluripotency, mESCs were cultured in the presence of 1000 U/mL leukemia inhibitory factor (LIF, Chemicon, Billerica, MA) and passaged upon reaching confluence every third day. Cells were maintained in a humidified incubator with 5% CO₂ at 37 °C.

Hazenbiller O., Duncan N.A, & Krawetz R.J. (2017). Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide. BMC Cell Biology, 18, 32.

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Culturing Human Umbilical Vein Endothelial and THP-1 Monocytic Cells

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HUVECs, primary human umbilical vein endothelial cells, obtained from a pool of donors, were purchased from Clonetics (Lonza, Switzerland) and cultured in endothelial basal medium (EBM-2, CC-3156, Lonza) supplemented with SingleQuot Bullet Kit (CC-4176, Lonza) containing 0.1% human recombinant epidermal growth factor (rh-EGF), 0.04% hydrocortisone, 0.1% vascular endothelial growth factor (VEGF), 0.4% human recombinant fibroblast growth factor (rh-FGF-B), 0.1% insulin-like growth factor-1 with the substitution of arginine for glutamic acid at position 3 (R3- IGF-1), 0.1% ascorbic acid, 0.1% heparin, 0.1% gentamicin and amphotericin-B (GA-1000), and 2% fetal bovine serum (FBS). The cells were seeded at a density of 5000/cm² in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis MO, USA).
Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and maintained in RPMI-1640 medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM and with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine (all from Euroclone, Milano, Italy). The cells were seeded at a density of 2 × 10⁵ cells/ml in T75 flasks.

Matacchione G., Gurău F., Silvestrini A., Tiboni M., Mancini L., Valli D., Rippo M.R., Recchioni R., Marcheselli F., Carnevali O., Procopio A.D., Casettari L, & Olivieri F. (2021). Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and β-caryophyllene association on human endothelial and monocytic cells. Biogerontology, 22(3), 297-313.

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H9C2 Cell Culture Methodology

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H9C2 cells (rat embryonic ventricular myocytes) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were routinely cultured in complete DMEM media containing 5 mM glucose and L-glutamine (Gibco Laboratories, USA) and supplemented with 10% fetal bovine serum (Gibco Laboratories, USA) and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin (Gibco Laboratories, USA)). The cell lines were cultured in T75 flasks (Sigma) at 37°C in a humidified atmosphere, with 5% CO₂. Media were replaced every 2 to 3 days and cells were split when they approximately reach 80% confluence. H9C2 in logarithmic growth phase were trypsinized and seeded in 6- or 96-well plates (Sigma) for follow-up experiments.

Zhang Y., Su W., Zhang Q., Xu J., Liu H., Luo J., Zhan L., Xia Z, & Lei S. (2018). Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCβ2 Activation and Improving Mitochondrial Quality. Journal of Diabetes Research, 2018, 9502895.

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Isolation and Expansion of Mesenchymal Stromal Cells

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MSCs were obtained from the marrow of two different living donors (Lonza, Walkersvile, MD) and from a cadaveric donor. Living donor BM was either directly cultured or was first processed using a ficoll gradient-density sedimentation to obtain a mononuclear fraction (MNC) before plating. Organ donor marrow was obtained as previously described (20 (link)) and was subjected to a ficoll gradient-density sedimentation to obtain MNCs that were stored in frozen aliquots that were thawed and then plated.
Whole BM or MNCs were inoculated into T-75 flasks (Corning Costar, Sigma Aldrich, St. Louis, MO) containing basal medium supplemented with 10% FBS, 10% PL-S, or 10% PL-P. In cultures supplemented with PL-P, 2 IU/mL of heparin (APP Pharmaceutical, Schaumburg, IL) was added to preclude clot formation. After 48 hours of incubation, non-adherent cells were removed, fresh medium was added and the medium was changed every 3–4 days until cells reached 80–90% confluence. Cells were detached with 0.25% trypsin (HyClone, Logan, UT), re-seeded and expanded for two more passages (P1 and P2). At each passage, cell counts and viabilities were performed. P2 cells were analyzed for MSC cell surface markers per ISCT guidelines (21 (link)) and were frozen in aliquots of 5 × 10⁶ MSC for gene expression analysis.

Pierce J., Benedetti E., Preslar A., Jacobson P., Jin P., Stroncek D.F, & Reems J.A. (2017). Comparative Analyses of Industrial Scale Human Platelet Lysate (PL) Preparations. Transfusion, 57(12), 2858-2869.

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Characterization of Head and Neck Cancer Cell Lines

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Two head and neck cancer cell lines were use in this study. Both were purchased through Merck Millipore (Darmstadt Germany).
A. UM-SCC-1 was isolated from a recurrent squamous cell carcinoma in the mouth floor of a 73-year-old male. The cell line originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is negative for the Human Papilloma Virus (HPV).
B. UM-SCC-47 is also a squamous cell carcinoma but derived from a primary tumour of the lateral tongue. This cell line also originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is HPV positive for HPV type 16. Oncogenicity is conferred through the expression of viral oncoproteins E6 and E7 [35 (link)].
Cell lines were cultured in T75 flasks (Sigma-Aldrich^® Darmstadt DE) as a monolayer using RPMI 1640 medium (Sigma-Aldrich^® Darmstadt DE) supplemented with 10% foetal calf serum (FCS), 10 mM HEPES, 12.5 μg/ml penicillin and 16 μg/ml gentamycin. Cell flasks were incubated in a humidified atmosphere at 37°C containing 5% CO₂ and passaged after reaching exponential growth prior to confluency. Both cell lines were tested for the presence of mycoplasma (biotool.com B3903, Madrid ES) and found to be negative.

Reid P., Wilson P., Li Y., Marcu L.G., Staudacher A.H., Brown M.P, & Bezak E. (2017). In vitro investigation of head and neck cancer stem cell proportions and their changes following X-ray irradiation as a function of HPV status. PLoS ONE, 12(10), e0186186.

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