293FT cells (Invitrogen, R70007) were maintained in DMEM high-glucose media (Gibco) supplemented with 10% FBS (Ausbian). U-2 OS cells (ATCC, HTB-96) were maintained in McCoy’s 5 A (Modified) Medium (Gibco) supplemented with 10% FBS (Ausbian). Human adult fibroblasts were derived from skin biospies of two 35-year-old healthy male individuals with approval for collection and use of human samples by institutional ethical committees of Eye & ENT Hospital, Fudan University (2020035, 2021007-1). Human skin biopsy tissues were cut into about 1 mm × 1 mm × 1 mm pieces, and placed on 60 mm dishes (Corning) precoated with 15 µg/cm2 collagen type I from rat tail (Sigma-Aldrich) in 6 ml DMEM high-glucose media (Gibco) supplemented with 10% FBS (Ausbian) and 5 ng/ml bFGF (PeproTech), and put in the 37 °C incubator. After 3 weeks’ incubation, fibroblasts that migrated out of the tissues were passaged to new collagen-I-coated dishes.
60 mm dishes
Manufactured by Corning
Sourced in United States
The 60-mm dishes are a type of laboratory equipment used for cell culture applications. They provide a standardized and consistent surface area for the growth and observation of cells in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Tb green premix ex taq, by Takara Bio (2 mentions)
Linegene 9600 plus machine, by Bioer (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Pancreatin, by Merck Group (2 mentions)
Dmem high glucose media, by Thermo Fisher Scientific (1 mentions)
Collagen type 1 from rat tail, by Merck Group (1 mentions)
293ft cell, by Thermo Fisher Scientific (1 mentions)
U2os cell, by American Type Culture Collection (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a modified medium, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by Thermo Fisher Scientific (1 mentions)
Pei max, by Polysciences (1 mentions)
Gammacell 1000 elite, by Nordion (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Us dialyzed fbs, by GE Healthcare (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
29 protocols using 60 mm dishes
1
Culturing Human Adult Fibroblasts
2
Quantifying UBE2T Expression via RT-PCR
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Cells were incubated in 60 mm dishes (Corning) and Trizol (Invitrogen) was added at 70% density after PBS washing followed RNA isolation according to Trizol introductions. After concentration detection of total RNA extraction, 3 μg RNA was pipetted for RT-PCR (Applied Biosystems, Thermal Cycler) of UBE2T using the primers: forward primer 5′-CAAATATTAGGTGGAGCCAACAC-3′ and reverse primer 5′-TAGATCACCTTGGCAAAGAACC-3′, and agarose gel electrophoresis followed at 120 V for 30 min.
3
Colony Formation Assay for Renal Cancer
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Briefly, the transfected 786-O and A498 cells were seeded into 60-mm dishes (Corning, NY, USA) at a density of 2 × 103 cells/well, respectively, followed by incubation at 37 °C for 10–14 days. 769-P cells were seeded into 60-mm dishes at a density of 2 × 103 cells/well. After 24 h, cells were treated with MLN8237 (10 μM), SZL P1-41 (20 μM), or MLN8237 + SZL P1-41 for 48 h, which was then removed and replaced by complete medium, followed by incubation at 37 °C for 10–14 days. The colonies were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet at room temperature for 30 min, and then counted [38 (link)]. Each experiment was conducted in triplicate.
4
Biofilm RNA Extraction and Quantification
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Bacteria were grown in 1/10 TSB medium at 28°C for 24 h in 60 mm dishes (Corning). The supernatant was discarded and the bacteria accumulated in the biofilms under the dishes were scraped. The total RNA was extracted using a Bacterial RNA Kit (Omega). The cDNA was synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (Takara) and quantified via TB Green Premix Ex Taq (Takara). The gene transcript levels were tested in triplicate for real-time PCR in a Linegene 9600 Plus machine (Bioer). Primer pairs of Qgyrb-F/R, QcsgD-F/R, QcsgA-F/R, QadrA-F/R, and QbcsA-F/R (Table 3 ) were used for the mRNA detection of gyrB, csgD, csgA, adrA, and bcsA, respectively. The target genes' mRNA levels were normalized to the gyrB mRNA levels (2−ΔΔCt) (30 (link)).
5
HeLa Cell Transfection with PEI
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HeLa cells were seeded at 1.5 × 105 cells/mL in 60 mm dishes (Corning, Corning, NY, USA) 24 h prior to performing the experiment. Polyethylenimine (PEI) “Max” (Polysciences, Warrington, PA, USA)-mediated transfection was carried out using the appropriate constructs. Typically, 5 μg of DNA and 0.02 mL of PEI (1 μg/μL) were mixed in 0.4 mL of a 150 mM NaCl solution. After incubation for 20 min at 25 °C, the mixture was added to the cells. The cells were incubated for 24 h post transfection.
6
PV-10 Radiosensitization Assay
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Cells were seeded at 5×104 in 60 mm dishes (Corning Incorporated) and incubated for 24 hours. The cells were then treated with either PBS (vehicle control) or 50 µM PV-10 and cultured for 4 hours, protected from light. Cells were irradiated with 0.5, 1, or 2 Gray (Gy) using a Gammacell 1,000 Elite (MDS Nordion, Kanata, ON, Canada) and cultured for 92 hours in the dark. Treatments were run in triplicate. Dishes were washed twice with PBS and cell viability was analyzed by alamar blue, as described for cytotoxicity assays.
7
Tryptophan Starvation of HeLa Cells
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Trp sarvation of HeLa cells were induced as previously reported [38 (link)]. In detail, HeLa cells were seeded in 60 mm dishes (Corning) and incubated for 24 h. Cells were washed (3×) in PBS and incubated for 24 h in amino acid-free DMEM medium (high glucose) (#048-33575; FUJIFILM Wako Pure Chemical Corp.) supplemented with 10% (v/v) US dialyzed FBS (GE Healthcare Life Sciences), 2 mM Gln, and amino acids (0.4 mM arginine, 0.2 mM cysteine, 0.4 mM glycine, 0.2 mM histidine, 0.8 mM isoleucine, 0.8 mM leucine, 0.8 mM lysine, 0.2 mM methionine, 0.4 mM phenylalanine, 0.4 mM serine, 0.8 mM threonine, 0.4 mM tyrosine, 0.8 mM valine). Note, with the exception of Trp, the concentration of each of the amino acids corresponds to that of standard DMEM (#10313-021; ThermoFisher Scientific).
8
Lipofectamine 3000 siRNA Transfection
9
Colony Formation Assay for Cell Lines
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Briefly, the transfected H1299 and H358 cells were seeded into 60-mm dishes (Corning, NY, USA) in triplicate at a density of 2000 cells/well, followed by incubation at 37 °C for 14 days. The colonies were fixed with 4% paraformaldehyde for 15 min, stained with crystal violet at room temperature for 30 min, and then counted [30 (link)].
10
Forskolin Modulates BeWo Cell Gene Expression
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BeWo cells were cultured until 50% confluency on 60 mm dishes (Corning). At this point, the cells were either incubated with Forskolin (20 mM) or DMSO as a control. Cells were harvested after 24 h, 48 h, and 72 h. Changes in gene expression were analysed by quantitative PCR (qPCR) and Immunoblotting.
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