10 cm dishes

Manufactured by Corning

Sourced in United States

The 10 cm dishes are sterile, single-use culture vessels made of polystyrene. They provide a circular growth surface area of approximately 78.5 square centimeters. These dishes are commonly used for various cell culture applications in laboratories.

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40 protocols using 10 cm dishes

Investigating LAB-SCS Effects on Lipid Secretion

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A monolayer of the HepG2 cell line (BCRC 60025) was obtained from BCRC. The cells were maintained in Eagle's minimum essential medium with 10% foetal bovine serum and 50 U/mL penicillin-streptomycin solution (Gibco, Grand Island, NY) at 37°C and 5% CO₂. HepG2 cells were subcultured in 10 cm dishes (Corning Costar) to 80% confluence. Since HepG2 cells are highly dependent on a high concentration of exogenous fatty acids to maintain an adequate supply of lipids for lipoprotein assembly [20 (link)], we investigated the LAB-SCS effect on apo B and triglyceride (TG) secretion in cells incubated with oleic acid (OA). OA was provided as a complex with bovine serum albumin (BSA). The OA/BSA molar ratio was 8 : 1, and the concentrations were 0.81 mmol/L for OA and 0.1 mmol/L for BSA [21 (link), 22 (link)].
HepG2 cells (10⁵ cell/mL) were incubated in 24-well plates (Corning Costar) with or without 100 μL LAB-SCS per well overnight in an incubator (37°C, 5% CO₂). At various time points (12, 24, 36, and 48 h), cells were harvested from the plate, lysed in 1% Triton-X 100, and then sonicated for 15 sec. The lysates were centrifuged and the supernatants were collected to measure the apo B and TGs concentrations using an apo B ELISA kit and a glycerol-3-phosphate oxidase and phenol 4-aminoantipyrine peroxidase method (GPO-PAP) kit (RANDOX, Antrim, UK).

Tsai C.C., Lin P.P., Hsieh Y.M., Zhang Z.Y., Wu H.C, & Huang C.C. (2014). Cholesterol-Lowering Potentials of Lactic Acid Bacteria Based on Bile-Salt Hydrolase Activity and Effect of Potent Strains on Cholesterol Metabolism In Vitro and In Vivo. The Scientific World Journal, 2014, 690752.

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Establishment and Characterization of Endometrial Adenocarcinoma Cell Lines

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The human endometrial adenocarcinoma cell line, the Ishikawa cell line [92 (link)], was obtained from American Type Culture Collection (ATCC) (Manassas, VA) [93 (link)]. The primary tumor-derived endometrial adenocarcinoma cell lines originated from patients with Stage IC Grade 3 (ACI-181), Stage IIB Grade 2 (ACI-52), and Stage IIIC Grade 2 (ACI-80) International Federation of Gynecology and Obstetrics (FIGO) staging (gift from Dr. Risinger, Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids 49503, MI, USA) (Supplementary Table 1). All cell lines used in this study are identified as having endometrioid histologic characteristics and were grown-maintained as previously described [74 (link), 75 (link)]. In brief, cells were grown in phenol red free-DMEM (Cellgro-Mediatech, Inc., Manassas, VA, USA) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and 2 mM L-Glutamine (Quality Biological Inc., Gaithersburg, MD, USA). These cells were maintained at 37°C with humidified atmosphere of 5% CO₂ until 90–100% confluence was reached. Cells were subsequently passaged in a 1:5 ratio into 10-cm dishes (Costar, Corning, NY, USA).

Cho-Clark M.J., Sukumar G., Vidal N.M., Raiciulescu S., Oyola M.G., Olsen C., Mariño-Ramírez L., Dalgard C.L, & Wu T.J. (2021). Comparative transcriptome analysis between patient and endometrial cancer cell lines to determine common signaling pathways and markers linked to cancer progression. Oncotarget, 12(26), 2500-2513.

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Curcumin Modulates Protein Interactions

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After transfection and curcumin treatment, cell protein was extracted and protein concentration was measured using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Protein lysates were loaded and separated on 10% SDS-polyacrylamide gels, then transferred 2 h in cold transfer buffer. Membranes were blocked in 5% fat-free milk for 2 h at room temperature and then incubated with primary antibodies and secondary antibodies as described previously.33 (link) Protein bands were visualized using enhanced chemiluminescence detection reagent (Thermo Fisher Scientific) and Image Lab™ software 4.1 (Bio-Rad Laboratories, CA, USA). The protein bands were analyzed as a percentage of β-actin levels.
For Co-IP assays, cells were cultured in 10 cm dishes (Corning) and divided into untreated (mock) and curcumin-treated groups for 48 h. The cells were lysed in RIPA lysis buffer (Beijing Solarbio Science & Technology) on ice for 30 min, and precleared with protein-G agarose beads (Sigma) for 4 h at 4°C. Lysates were then incubated overnight at 4°C with anit-Gli1 or anti-β-catenin primary antibodies, on a rotating wheel. The next day, antibody-antigen complexes were analyzed by Western blotting as described.

Zhang X., Zhang C., Ren Z., Zhang F., Xu J., Zhang X, & Zheng H. (2020). Curcumin Affects Gastric Cancer Cell Migration, Invasion and Cytoskeletal Remodeling Through Gli1-β-Catenin. Cancer Management and Research, 12, 3795-3806.

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HeLa and HEK293T Cell Culture and Transfection

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HeLa and HEK293T cells (ATCC, Manassas, VA) were cultured in complete DMEM (CDMEM) that consisted of Dulbecco’s Modified Eagle’s Medium (DMEM) (112-319-101, Quality Biological, Gaithersburg, MD) supplemented with 10% fetal bovine serum (35–011-CV, Corning, NY), 50 U/mL penicillin, 50 μg/mL streptomycin (30002-CL, Corning), and MycoZap Plus-CL (VZA-2011, Lonza, Basel, Switzerland) at 37°C and 5% CO₂. For immunofluorescence microscopy, transfections were performed using 24-well plates (Corning) with 0.1–0.5 μg plasmid and 1μL Lipofectamine 2000 (11668019, Thermo Fisher scientific, Waltham, MA) according to the manufacturer’s protocol. Transfection medium was replaced with CDMEM after 1 h. Cells were analyzed ~24 h after transfection. For FKBP-FRB dimerization experiments (Figure 3H–I), 1 h after transfection, the medium was replaced with CDMEM supplemented with 0.5 μM A/C heterodimerizer (Takara Bio Inc., Shiga, Japan). For co-immunoprecipitation experiments, cells were grown in 10-cm dishes (Corning) and transfections were performed using 1–8 μg plasmid. Cells were kept in the transfection medium until they were harvested.

Keren-Kaplan T, & Bonifacino J.S. (2020). ARL8 Relieves SKIP Autoinhibition to Enable Coupling of Lysosomes to Kinesin-1. Current biology : CB, 31(3), 540-554.e5.

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Restoring CypA and CypB Expression in KD Cells to Study FCoV Infection

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KD-CypA-fcwf cells and KD-CypB-fcwf cells were plated on 10 cm dishes (Corning) and transfected with 5 µg DNA of pEF-Myc-CypA and pEF-Myc-CypB, respectively, using the X-tremeGENE HP DNA Transfection Reagent according to the manufacturer’s protocol. The transfected cells were selected with both 6 µg ml⁻¹ blasticidin and 6 µg ml⁻¹ puromycin at 24 h after transfection. The individual cell colonies selected with blasticidin and puromycin were cloned and analysed for expression of the target proteins by Western blot. Those KD cells restored with CypA or CypB were infected with FCoV 79–1146 at a m.o.i. of 1 p.f.u. per cell to study the effects on FCoV infection before cells were collected for Western blot and RT-qPCR analyses at 20 h p.i.

Tanaka Y., Sato Y, & Sasaki T. (2017). Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B. The Journal of General Virology, 98(2), 190-200.

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Analyzing GR Homodimerization in U2OS Cells

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U2OS cells were purchased from ATCC (Lot# 58078676) and were maintained in McCoy’s 5 A modified medium (Gibco Life Technologies, UK) supplemented with 10% charcoal-stripped fetal bovine serum. FBS (Gibco Life Technologies, UK) was stripped overnight using 5% charcoal.
U2OS cells on the 6-well plate (Thermo Fisher Scientific) were transfected with 5 μg total plasmid DNA and ViaFect (Promega). For the determination of the dissociation constant for GR homodimerization, 5 μg pEGFP-GR/WT, its mutants, or 0.1 μg pEGFP and 4.9 μg empty vector were transfected. For elucidating the transcriptional activity in single cells, 1 μg pEGFP-GRs, 4 μg pMMTV-mKO2, and 0.1 μg pTagRFP675 were transfected. For in vitro DNA binding analysis by FCCS and electrophoretic mobility shift assay, U2OS cells on 10 cm dishes (Corning) were transfected with 15 μg pEGFP-GRs by ViaFect. At 6 h after the transfection, the cell culture medium was exchanged with fresh medium, and the U2OS cells were then cultured for 18 h.
To analyze single cells, individual transfected U2OS cells were seeded into microwells on the surface of polydimethylsiloxane (PDMS) chips (Fig. 1(a))^{14 (link)}. The cell suspension with 3.0 × 10⁴ (cells/mL) was poured on the PDMS chips after washing with detergent water. Cells were incubated to adhere cells to PDMS chips for 4 h.

Oasa S., Mikuni S., Yamamoto J., Kurosaki T., Yamashita D, & Kinjo M. (2018). Relationship Between Homodimeric Glucocorticoid Receptor and Transcriptional Regulation Assessed via an In Vitro Fluorescence Correlation Spectroscopy-Microwell System. Scientific Reports, 8, 7488.

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Isolation and Cryopreservation of Amniotic Membrane

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This experiment was performed in 10 cm dishes (Corning, USA) in a biological safety cabinet. AM was treated with 100 U/mL of penicillin and 100 μg/mL of streptomycin (Thermo Fisher Scientific, USA) for 30 min and rinsed with sterile saline. Thereafter, it was treated with 0.1% Triton X-100 (Sigma-Aldrich) and incubated in a shaking incubator at 37°C for 36 h. AM was rinsed with saline and treated with 2.5% trypsin-EDTA (Thermo Fisher Scientific) at 37°C for 4 h. A cell scraper was used to separate the epithelial cell layer and the spongy layer. Tissues were cut into 1 cm × 2 cm fragments. A portion of the FAAM was cryopreserved in 0.1% glycerol and stored at -20°C.

Wang S., Shi C., Cai X., Wang Y., Chen X., Han H, & Shen H. (2021). Human Acellular Amniotic Matrix with Previously Seeded Umbilical Cord Mesenchymal Stem Cells Restores Endometrial Function in a Rat Model of Injury. Mediators of Inflammation, 2021, 5573594.

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Feeder-free Expansion of Human Pluripotent Stem Cells

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hPSCs were cultured in 125-mL disposable spinner flasks (Corning, VWR) on a nine-position stir plate (Dura-Mag) at a speed of 65 rpm, in a 37°C incubator with 5% CO₂, which is a higher speed than previously reported (Rigamonti et al., 2016 (link)). Before adaption to spinner flask culture, hPSCs were expanded in 10-cm dishes (Corning) until they reached confluence. Cells were dissociated using Accutase (Innovative Cell Technologies) for approximately 5 min at room temperature or until colonies detached from the plate. Cells were counted using a Bio-Rad automated cell counter. Forty million individual hPSCs were seeded into a spinner flask in 120 mL of StemFlex medium supplemented with 10 μM ROCK inhibitor Thiazovivin (STEMCELL Technologies). Spheres formed spontaneously, and, after 48 h, approximately half of the culture medium was replaced. The cells were maintained as undifferentiated pluripotent spheres in spin culture, with medium changes every other day until the spheres were approximately 500 μm (organoid area was measured using Nikon software in brightfield).

Ahfeldt T., Ordureau A., Bell C., Sarrafha L., Sun C., Piccinotti S., Grass T., Parfitt G.M., Paulo J.A., Yanagawa F., Uozumi T., Kiyota Y., Harper J.W, & Rubin L.L. (2020). Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons. Stem Cell Reports, 14(1), 75-90.

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Protein Co-Immunoprecipitation Assay

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HEK293T cells were grown to an appropriate density in 10 cm dishes (Corning, USA). Afterwards, the cells were co-transfected with 5 μg Myc-tagged protein expression plasmid and 5 μg FLAG-tagged plasmid (pCMS-FLAG as a control). Twenty-four hours after transfection, the cells were harvested and lysed using a cell lysis buffer (Beyotime, China). Next, 20 μL of cell lysate was isolated as an input sample to detect protein expression. An appropriate amount of anti-FLAG M2 magnetic beads (Sigma-Aldrich) was added to the remaining lysate samples. The mixture was incubated at 4°C for > 2 h with gentle shaking, after which the beads were rinsed 3 times with cell lysis buffer. Twenty microliters of input samples and rinsed magnetic beads were then mixed with 2× protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer (Takara) at 100°C under denaturation for 5 min. Western blot assays were employed for target protein examination using an anti-Myc antibody (Abbkine Scientific Co., Ltd., Wuhan, China) and an anti-FLAG antibody (Sigma).

Liu W., Ma J., Chen J., Huang B., Liu F., Li L., Fan N., Li F., Zheng Y., Zhang X., Wang X., Wang X., Wei L., Liu Y., Zhang M., Han Y, & Wang X. (2023). A novel TBK1/IKKϵ is involved in immune response and interacts with MyD88 and MAVS in the scallop Chlamys farreri. Frontiers in Immunology, 13, 1091419.

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Mammalian and Insect Cell Culture Protocol

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Most chemicals and molecular biology reagents were purchased from Sigma-Aldrich unless mentioned otherwise. HEK-293 cells (ATCC; cat. no. CRL-3216) were maintained at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (Gibco; cat. no. 12800-017) supplemented with 10% FBS (Gibco; cat. no. 10270-106) and 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Gibco; cat. no. 15140-122). Cells were cultured in 10 cm dishes (Corning; cat. no. 430167) at 37 °C under 5% CO₂ and passaged at 70 to 80% confluency using 0.05% trypsin-EDTA for detachment. Sf9 cells (Expression Systems; cat. no. 94-001 F) were maintained as suspension cultures in ESF 921 media (Expression Systems; cat. no. 96-001-01). Lauryl Maltose Neopentyl Glycol (LMNG) was purchased from Anatrace (cat. no. NG310).

Baidya M., Chaturvedi M., Dwivedi-Agnihotri H., Ranjan A., Devost D., Namkung Y., Stepniewski T.M., Pandey S., Baruah M., Panigrahi B., Sarma P., Yadav M.K., Maharana J., Banerjee R., Kawakami K., Inoue A., Selent J., Laporte S.A., Hébert T.E, & Shukla A.K. (2022). Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody. Nature Communications, 13, 4634.

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