Tissue culture flasks

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Tissue culture flasks are laboratory containers designed for the in vitro cultivation of cells, tissues, or organisms. They provide a controlled environment for cell growth and experimentation, facilitating the study and manipulation of biological samples.

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Lab products found in correlation

45 protocols using tissue culture flasks

Propagation of Human Coronavirus 229E

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MRC-5 embryonic lung fibroblast cells (ATCC^® CCL-171^TM) and Hep G2
cells (ATCC HB-8065^TM) were obtained from the American Type Culture Collection.
Both cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10%
heat inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin and expanded in
75 cm² tissue culture flasks (BD Falcon) to 80%–85% confluency. All cells
were cultured at 37 °C in 5% CO₂ in a humidified incubator, and FBS was
inactivated at 56 °C for 30 min. The human coronavirus 229E (ATCC VR-740^TM) was
propagated in MRC-5 cells. Briefly, MRC-5 cells (75%–80% confluent) cultured in T-75
flasks were infected with human coronavirus 229E (HCoV-229E) in the serum-free EMEM for 2
h, and without removing the initial inoculum, the medium was supplemented with 2%
FBS-containing EMEM for a final volume of 10 ml and then incubated at 33 °C for 3 days.
The cells were freeze-thawed twice, harvested, and centrifuged at 10 000 × g for 10 min at
4 °C. The cell supernatant was collected, filtered through a syringe filter of
0.22 μm pore size, aliquoted, and stored at −80 °C. The virus
preparation used in this study had a titer of 4.48 × 10⁴ TCID₅₀/ml,
as determined by the Reed and Muench method.⁴⁷ (link)

Delumeau L.V., Asgarimoghaddam H., Alkie T., Jones A.J., Lum S., Mistry K., Aucoin M.G., DeWitte-Orr S, & Musselman K.P. (2021). Effectiveness of antiviral metal and metal oxide thin-film coatings against human coronavirus 229E. Apl Materials, 9(11), 111114.

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Activation of Molt-3 T cells and HCAEC

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Molt-3 T cells were obtained from the American Type Culture Collection (Rockville, MD). They were maintained using RPMI1640 media containing 100 μg/l of penicillin/streptomycin along with 10% heat-inactivated fetal bovine serum (FBS). Tissue culture flasks (75-cm²; Falcon, Fisher, Pittsburgh, PA) were used to culture T cells at 37°C under a saturating humidified atmosphere of 95% air and 5% CO_2. Phorbol 12-myristate-13-acetate or PMA (0.2 μM; Sigma, St. Louis, MO) was used to activate these cells. HCAEC (Clonetics, San Diego, CA) were propagated and maintained using medium provided by the vendor in Tissue culture flasks (75-cm²; Falcon, Fisher, Pittsburgh, PA). HCAEC were activated with 20 ng/ml of TNF-α (Millipore Sigma) for 24 h.

Yusuf-Makagiansar H., Yakovleva T.V., Weldele M., Mahadik R, & Siahaan T.J. (2023). Evaluation of LFA-1 Peptide-Methotrexate Conjugates in Modulating Endothelial Cell Inflammation and Cytokine Regulation. Medical research archives, 11(2), 3534.

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Excretory-Secretory Antigen Preparation from Toxocara canis Larvae

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The excretory–secretory antigen of T. canis was prepared according to the method of de Savigny (1975). Larvae were cultivated in RPMI 1640 (Sigma-Aldrich, Hamburg, Germany) medium modified with 20 mM HEPES and l-glutamine, supplemented with penicillin/streptomycin (100 IU, 100 μg/ml) (Sigma-Aldrich, Hamburg, Germany). Larvae were maintained in sterile 25-ml tissue culture flasks (Falcon, Durham, USA) at a concentration of 10³ larvae/ml and incubated continually for a long term at 37°C under a 5% C0₂ and 95% atmosphere humidity. The culture medium was replaced at 5-day intervals after assessing the viability of larvae. Starting at the third week, the collected pooled medium (from 3 weeks) was concentrated in 3000 MWCO VIVASPIN tubes (Sartorius, Goettingen, Germany) at 4000 g for 100 min at 4°C. The protein concentration of the larval antigen was measured using the Bradford protein assay (Bio-Rad Laboratories, München, Germany).

Kołodziej-Sobocińska M., Dvorožňáková E., Hurníková Z., Reiterová K, & Zalewski A. (2020). Seroprevalence of Echinococcus spp. and Toxocara spp. in Invasive Non-native American Mink. Ecohealth, 17(1), 13-27.

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Enrichment of Activated NK Cells

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Spleens were removed aseptically from C57BL/6 and CD45.1 congenic B6.SJL-Ptprca Pep3b/BoyJ mice and a single-cell suspension was prepared in RPMI-1640. Erythrocytes were lysed by incubation with ammonium chloride–potassium buffer at room temperature for 3 min and the spleen cells were subsequently washed twice in RPMI-1640. CD3 and B220 positive cells were magnetically removed following incubation of the cell culture with rat anti-CD3 and rat-antiB220 antibody and subsequently with anti-rat coated magnetic beads (Dynal Biotech, Lake Success, NY, USA). The CD3/B220-depleted cells were resuspended in fresh CM containing 6,000 IU/ml rhIL-2 (kindly provided by Novartis Pharma AG, Basel, Switzerland) to a final concentration of 1×10⁵ cells/ml and cultured in tissue culture flasks (Falcon, B&D, Franklin Lakes, NJ, USA) at 37°C in an atmosphere of 5% CO₂. Fresh CM containing 6,000 IU/ml IL-2 was added every 2–3 days as needed. After 5–7 days of culture, non-adherent cells and adherent cells were harvested after a brief treatment with 0.02% EDTA and washed twice in RPMI-1640 before use. Routinely, on day 5 of culture, the A-NK cells were >95% CD45.1+ (or CD45.2+), >95% Thy1.2+, >95% asGM1+, >90% NK1.1+, >90% NKp46+ <2% CD8+, <2% CD4+.

Goding S.R., Yu S., Bailey L.M., Lotze M.T, & Basse P.H. (2016). Adoptive Transfer of Natural Killer Cells Promotes the Anti-Tumor Efficacy of T Cells. Clinical immunology (Orlando, Fla.), 177, 76-86.

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HUVEC Response to Hyperglycemia

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Human umbilical vein cells (HUVECs) were obtained from Life Technologies (ThermoFisher, Waltham MA), cultured in endothelial growth media (EBM-2 BulletKit, Clonetics LONZA, Walkersville MD), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies^™, Carlsbad, CA) under standard cell culture conditions (37°C and 5% CO₂). After reaching ~90% confluence on the 3rd passage, cells were seeded into tissue culture flasks (Falcon, Corning NY, USA) and incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (control cells, normoglycemic condition) for 48 hours. All experimental conditions contained 25mM D-manitol (VWR Analytical, Radnor PA) to control for osmolality [13 (link), 14 (link)]. After the 48-hour incubation period, cells and supernatant for both conditions were collected for analysis.

Bammert T.D., Hijmans J.G., Reiakvam W.R., Levy M.V., Brewster L.M., Goldthwaite Z.A., Greiner J.J., Stockelman K.A, & DeSouza C.A. (2017). HIGH GLUCOSE DERIVED ENDOTHELIAL MICROPARTICLES INCREASE ACTIVE CASPASE-3 AND REDUCE MicroRNA-Let-7a EXPRESSION IN ENDOTHELIAL CELLS. Biochemical and biophysical research communications, 493(2), 1026-1029.

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Immunofluorescence Microscopy Protocol

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Tissue culture flasks (Falcon), tissue culture media, trypsin, and 100× antibiotic-antimycotic solution (Cellgro, Mediatech, Inc) were purchased from Fisher Scientific Inc (Springfield, NJ). Triazol reagent and transfection reagent were purchased from Invitrogen Inc (Carlsbad, CA). For Western blot detection, Super Signal West Dura Extended Duration Substrate (PIERCE, Rockford, IL) was used. For immunofluorescence microscopy, Alexa Fluor 488 and 596 conjugated secondary antibodies and Hoechst 33342 nuclear counter staining dye were purchased from Molecular Probes Inc (Eugene, Oregon). Primary antibodies, anti-lamin A (H-102, rabbit polyclonal IgG), were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA).

Capo-chichi C.D., Yeasky T.M., Smith E.R, & Xu X.X. (2016). Nuclear envelope structural defect underlies the main cause of aneuploidy in ovarian carcinogenesis. BMC Cell Biology, 17, 37.

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Culturing and Treating SW480 Cells

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SW480 cells were originally purchased from the Pasteur Institute of Iran (Tehran, Iran). RPMI 1640 medium (Gibco-BRL, Life technology, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, Life technology, Paisley, Scotland) and 1% penicillin-streptomycin (Gibco-BRL, Life technology, Paisley, Scotland) were used for growing of cells. Cells were seeded in tissue culture flasks (Falcon, Heidelberg, Germany) and retained in a humidified incubator with 37°C and 5% CO2. For the administration of drugs, AP and SP were purchased from Sigma-Aldrich. The stock solution of these drugs was made in sterile dimethyl sulfoxide (DMSO) and culture medium, respectively. After that, they were divided into aliquots and stored at -80°C until use. DMSO concentration was added less than 0.1% of the total volume and adjusted as an applied solution.

Ghahremanloo A., Javid H., Afshari A.R, & Hashemy S.I. (2021). Investigation of the Role of Neurokinin-1 Receptor Inhibition Using Aprepitant in the Apoptotic Cell Death through PI3K/Akt/NF-κB Signal Transduction Pathways in Colon Cancer Cells. BioMed Research International, 2021, 1383878.

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Viral Titer Determination for SIRV

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Cultures were started from frozen stocks in 2 mL of liquid DT medium for each of the S. islandicus host strains. When cultures turned cloudy, they were transferred into 20 mL of DT medium in tissue culture flasks (BD Falcon, Bedford, MA, USA) and incubated without shaking. For each culture, approximately 3 × 10⁸ cells were collected from mid-log cultures by low speed centrifugation, resuspended in 500 μL of DT medium, mixed with 100 μL of each dilution (10°–10⁻¹⁰), and plated on overlays of SY medium containing 0.1% yeast extract and 0.2% sucrose, as previously described [36 (link)], to determine viral titers. Dilutions were performed and plated in triplicate for each sample. SIRV stocks were diluted to 1 × 10⁷, 1 × 10⁶ and 1 × 10⁴ pfu (plaque forming units)/mL, and lawns of host cells were spotted with 10 μL of each of the virus dilutions and incubated at 78 °C. Lawns were monitored every 24 h for five days. All SIRVs were spotted on their susceptible isolation host as a positive control of infection. Three independent virus dilutions from the same original stock were spotted on three independent host cultures.

Bautista M.A., Black J.A., Youngblut N.D, & Whitaker R.J. (2017). Differentiation and Structure in Sulfolobus islandicus Rod-Shaped Virus Populations. Viruses, 9(5), 120.

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Isolation and Characterization of Bacterial Strains from Nematodes

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Bacterial strains were obtained by sonication of nematode hosts after surface sterilization. Strain identity was confirmed by analysis of 16S rRNA from whole-genome sequences (25 (link), 43 (link)) and average nucleotide identity (26 (link)). Bacterial strains were stored in lysogeny broth (LB) supplemented with 20% glycerol frozen at −80°C. Bacterial cultures were grown at 30°C in LB that had not been exposed to light, with aeration, or on LB agar with 0.1% pyruvate (45 (link)). S. feltiae nematode isolates used were obtained from the laboratories of Patricia Stock or Byron Adams (via Adler Dillman) and were verified through sequencing of the 12S and 28S genes (24 (link)). Nematodes were propagated through Galleria mellonella larvae (46 (link)). Axenic IJs were produced in vitro as previously described (47 (link), 48 (link)). Nematodes were stored at 25°C in 250-ml tissue culture flasks (BD Falcon, Franklin Lakes, NJ) at a density of 5 to 10 IJ/µl and a volume of less than 60 ml.

Murfin K.E., Lee M.M., Klassen J.L., McDonald B.R., Larget B., Forst S., Stock S.P., Currie C.R, & Goodrich-Blair H. (2015). Xenorhabdus bovienii Strain Diversity Impacts Coevolution and Symbiotic Maintenance with Steinernema spp. Nematode Hosts. mBio, 6(3), e00076-15.

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Establishing Bovine Fetal Fibroblast Cell Lines

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Bovine fetal fibroblast cell lines were established using a modification of a method described earlier17 . Briefly, bovine embryos flushed from the recipient cows were transported to the laboratory in Dulbecco's phosphate buffered saline solution (DPBS) with 16 μl/ml of antibiotic-antimycotic (Life Technologies, Grand Island, NY), and 8 μl/ml fungizone (Life Technologies). Embryos were rinsed in DPBS, the fetal membranes were removed and the whole embryo was disaggregated using a scalpel. Fibroblasts were separated by a standard trypsinization procedure using Tryp-LE (Life Technologies). Cells were seeded onto 25 mm tissue culture flasks (BD Falcon) in DMEM (Life Technologies) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 0.15 g/ml glutamine (Sigma), and 0.003% β-mercaptoethanol (Life Technologies). On day 4 of seeding, the cells were passaged and reseeded onto 250 mm tissue culture flasks. Cells were harvested and frozen in DMEM with 10% FCS and 10% DMSO (Sigma) once they were 80–90% confluent.

Kasinathan P., Wei H., Xiang T., Molina J.A., Metzger J., Broek D., Kasinathan S., Faber D.C, & Allan M.F. (2015). Acceleration of genetic gain in cattle by reduction of generation interval. Scientific Reports, 5, 8674.

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