Tissue culture flask

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Tissue culture flasks are laboratory equipment used for the in vitro cultivation of cells, tissues, or organisms. They provide a controlled environment for the growth and maintenance of cell cultures. These flasks are available in various sizes and shapes to accommodate different experimental needs.

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38 protocols using tissue culture flask

Skeletal Muscle FAPs Isolation and Culture

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Skeletal muscle tissue dissociation by enzymatic digestion and isolation of FAPs by FACS with anti-CD140a (PDGFRA)–APC (eBioscience, 17-1401-81) and anti-Ly6A/E (SCA-1)-v450 (BD Biosciences, 560653) was conducted as previously described (6 (link), 12 (link), 18 (link), 35 (link)), with the following modification: magnetic depletion of CD31⁺ and CD45⁺ cells prior to FACS was omitted, and CD31⁺ and CD45⁺ cells were detected with anti-CD31-bv711 (BD Biosciences, 740690; 1:800) and anti-CD45-bv711 (BD Biosciences, 563709; 1:500) antibodies. Sorting was performed on a FACSAria II (BD Biosciences) equipped with 405, 488, 561, and 633 nm lasers.
FACS-isolated FAPs were grown on tissue culture flasks (Nunc) in DMEM (Thermo Fisher Scientific, 11995065) with 50 U/mL penicillin and 50 μg/mL streptomycin (Pen/Strep; Gibco) and 20% premium FBS (Atlanta Biologicals, lot C19032) as previously described (6 (link), 12 (link)). FAPs were maintained at 37°C in a humidified atmosphere at 5% CO₂. Media were changed every other day. All experiments utilized FAPs that had undergone less than 7 population doublings (approximately 3 passages).

Lees-Shepard J.B., Stoessel S.J., Chandler J.T., Bouchard K., Bento P., Apuzzo L.N., Devarakonda P.M., Hunter J.W, & Goldhamer D.J. (2022). An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice. The Journal of Clinical Investigation, 132(12), e153795.

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Photoautotrophic Growth of Cyanobacteria

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Synechocystis sp. PCC6803 (WT) (not the glucose tolerant strain) and Synechococcus sp. PCC7942 were grown photoautrophically in BG-11 medium (Castenholz, 1988 ) at 30°C under 8 μE m⁻² s⁻¹ white light in tissue culture flasks (Nunc), with continuous shaking. For HL, cells were either incubated in BG-11 at 30°C under 600 μE m⁻² s⁻¹ white light or spotted onto BG-11 plates and similarly illuminated. E. coli strains used were DH5α and BW25113 (E. coli stock centre).

Bryan S.J., Burroughs N.J., Shevela D., Yu J., Rupprecht E., Liu L.N., Mastroianni G., Xue Q., Llorente-Garcia I., Leake M.C., Eichacker L.A., Schneider D., Nixon P.J, & Mullineaux C.W. (2014). Localisation and interactions of the Vipp1 protein in cyanobacteria. Molecular Microbiology, 94(5), 1179-1195.

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Isolation and Expansion of Adipose-Derived Stem Cells

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ASC isolation and expansion were performed at the Regenerative Medicine Laboratory at the William R. Pritchard Veterinary Medical Teaching Hospital, according to previously established protocols [17 (link)]. Briefly, ASCs were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Corning Life Sciences, Manassas, VA, http://www.cellgro.com), 10% FBS (HyClone Inc., Logan, UT, http://promo.gelifesciences.com), and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com) in tissue culture flasks (Nunc, Roskilde, Denmark, http://www.thermofisher.com) and incubated at 37°C in 5% carbon dioxide. Cells were passaged once they reached approximately 70% confluence. Fresh, expanded, early-passage cells were used for treatment (second or third passage) and the remaining cells were cryopreserved. For the subsequent dose (at 4 weeks after the first dose), an aliquot of first-passage cells were thawed and cultured expanded for 72 hours to regain cell viability and function prior to infusion, effectively using second- or third-passage cells. Cells are provided in glass vials to avoid plastic adherence while awaiting administration.

Arzi B., Mills-Ko E., Verstraete F.J., Kol A., Walker N.J., Badgley M.R., Fazel N., Murphy W.J., Vapniarsky N, & Borjesson D.L. (2015). Therapeutic Efficacy of Fresh, Autologous Mesenchymal Stem Cells for Severe Refractory Gingivostomatitis in Cats. Stem Cells Translational Medicine, 5(1), 75-86.

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Photoautotrophic Growth of Synechocystis Mutants

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Wild-type Synechocystis sp. PCC 6803 (glucose tolerant strain), M55, ΔhoxYH, ΔhoxE and the ΔhoxE over expressed strain (oxhoxE)^{12 (link),25 (link)} were grown photoautrophically in BG-11 medium³¹ at 30 °C. For our standard low-light (LL) conditions cells were grown under 8 μmol m^–2 s^–1 white light in tissue culture flasks (Nunc UK), with continuous shaking. For dark adapted conditions (DA) cells were grown at 30 °C in tissue culture flasks wrapped in foil for 5 days, before being spotted in low light onto BG-11 plates. High light (HL) cells were spotted onto BG-11 plates and illuminated for 10 min under 600 μmol m^–2 s^–1 white light. For anoxic conditions, catalase (500 U), glucose (5 mM), and glucose oxidase (30 U) were added to LL cells to make the medium totally anaerobic before being spotted onto BG-11 agar plates. E.coli strains used in this study were DH5α and BW25113 (E. coli stock centre). LL cultures were incubated for 1 h with 20 μM DCMU and 5 μM DBMIB. Lincomycin was added at 100 μg ml^–1 to DA cells prior to HL exposure.

Burroughs N.J., Boehm M., Eckert C., Mastroianni G., Spence E.M., Yu J., Nixon P.J., Appel J., Mullineaux C.W, & Bryan S.J. (2014). Solar powered biohydrogen production requires specific localization of the hydrogenase †Electronic supplementary information (ESI) available: Supplementary Fig. 1–12 and supplementary Table 1. See DOI: 10.1039/c4ee02502d Click here for additional data file.. Energy & Environmental Science, 7(11), 3791-3800.

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Culturing Colorectal Cancer Cell Lines

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The human colorectal adenocarcinoma cell line Caco2 is from ATCC-number HTB-37 and HT-29 is from ATCC-number HTB-38. The colorectal carcinoma cell line HCT116 was a generous gift from Dr. György Vereb, Department of Biophysics, University of Debrecen. Caco2 and HCT116 cells were cultured in RPMI-1640 medium supplemented with 1% antibiotic-antimycotic solution and 20% fetal bovine serum (GIBCO by Life Technologies, EU) in tissue culture flasks (Nunclon, Rochester, NY) at 37°C in 10% and 5% CO₂, respectively. HT-29 cells were cultured in RPMI-1640 medium supplemented with 1% antibiotic-antimycotic solution and 10% fetal bovine serum in 5% CO₂. Cell culture medium was replaced every 2-3 days and the cells were passaged when subconfluent.

Chatterjee A., Gogolak P., Blottière H.M, & Rajnavölgyi É. (2015). The Impact of ATRA on Shaping Human Myeloid Cell Responses to Epithelial Cell-Derived Stimuli and on T-Lymphocyte Polarization. Mediators of Inflammation, 2015, 579830.

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Cytotoxicity Evaluation of HeLa Cells

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The continuous human cell lines HeLa (epithelial cervical cancer cell line) (ATCC, Manassas, VA, USA) were used to investigate the cytotoxicity effect of new products. This adherent cell line was grown in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) foetal calf serum (FCS) (Gibco) and 2 mM l-glutamine (Sigma-Aldrich) in tissue culture flasks (Nunc, Roskilde, Denmark). It was sub-cultured twice a week and kept at 37 °C in a humidified and controlled atmosphere of 95% air and 5% CO₂.

Bouzina A., Bouone Y.O., Sekiou O., Aissaoui M., Ouk T.S., Djemel A., Mansouri R., Ibrahim-Ouali M., Bouslama Z, & Aouf N.E. (2023). In vitro antitumor activity, molecular dynamics simulation, DFT study, ADME prediction, and Eg5 binding of enastron analogues. RSC Advances, 13(28), 19567-19584.

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Cytotoxicity and Antioxidant Evaluation of Raji and HeLa Cells

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The Raji cells (human Burkitt’s lymphoma), harboring the latent form of EBV cycle [51 (link)], were obtained from the Institute Pasteur of Tunis. Hela cell line (epithelial cell line derived from cervical cancer) was supplied by ATCC (Manassas, VA, USA). Both cells were employed for cytotoxicity and antioxidant studies. Both cell lines were grown in RPMI 1640 medium (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS) and 2 mM l-glutamine in tissue culture flasks (Nunc). They were passed twice a week and kept at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Ben-Amor I., Gargouri B., Attia H., Tlili K., Kallel I., Musarra-Pizzo M., Sciortino M.T, & Pennisi R. (2021). In Vitro Anti-Epstein Barr Virus Activity of Olea europaea L. Leaf Extracts. Plants, 10(11), 2445.

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Mouse Fibroblast and Stem Cell Culture

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Mouse embryonic fibroblasts (MEFs) and transgenic MEFs (MEF Atg5 KO and MEF Bax/Bak DKO) were grown in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-Glutamine and 1% penicillin/streptomycin (Gibco, Invitrogen, Belgium). The MEF cells were passaged upon reaching 80% confluence and reseeded at a ratio of 1:5.
Mouse mesenchymal stem cells were maintained in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 10% horse serum, 1 mM sodium pyruvate and 2 mM L-Glutamine (Gibco, Incitrogen, Belgium). Cells were passaged when reaching nearly 80% confluence and reseeded at a density of 100,000 cells/flask in 75 cm² tissue culture flasks (Nunc, Belgium).

Manshian B.B., Munck S., Agostinis P., Himmelreich U, & Soenen S.J. (2015). High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations. Scientific Reports, 5, 13890.

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Cell Culture Protocol for Genetic Authentication

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All cell lines were obtained from the American Type Culture Collection ATCC and grown in Minimal Essential Medium (MEM) supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich ApS, Søborg, Denmark). DLD1 cells were a kind gift from Professor Lene Nejsum of Aarhus University (Aarhus, Denmark).
The cell cultures were maintained in a humidified incubator (5% CO₂/95% air atmosphere at 37 °C). Cells were plated into tissue culture flasks (Nunc, Roskilde, Denmark) and split every 3 days to maintain them at 70% confluency. Cells were harvested by trypsin treatment (0.1% Trypsin-EDTA solution, Sigma-Aldrich ApS, Søborg, Denmark) followed by two consecutive washes with 1 × PBS and stored at −80 °C as dried pellets until further analysis. All cell lines were genetically authenticated by the ATCC and mycoplasma tested by Eurofins Genomics (GATC service, Eurofins Genomics Ebersberg, Germany). Results of mycoplasma analysis were negative.

Tesauro C., Keller J.G., Gromova I., Gromov P., Frøhlich R., Erlandsen J.U., Andersen A.H., Stougaard M, & Knudsen B.R. (2020). Different Camptothecin Sensitivities in Subpopulations of Colon Cancer Cells Correlate with Expression of Different Phospho-Isoforms of Topoisomerase I with Different Activities. Cancers, 12(5), 1240.

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Culturing HT1080 Cells for Assays

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HT1080 cells were grown as monolayers in tissue culture flasks (Nunc, Denmark) in EMEM medium supplemented with 10% fetal bovine at 5% CO₂ and 37℃ in a humidified atmosphere. Cells were passaged thrice a week by treating with trypsin-EDTA, and then used for subsequent experiments. Once the cells adhered to the culture plate, the medium was replaced with EMEM medium containing 1% FBS, and the extract was added at various concentrations. The conditioned medium was collected after 24 h of incubation for subsequent analysis.

Kim J.H., Hwang G.H., Kim H.J., Jeon S, & Shin B.A. (2021). Acer mono Extract Inhibits Invasive Activities and G1/S Transition of HT1080 Fibrosarcoma Cells. Chonnam Medical Journal, 57(3), 185-190.

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