First branchial arch-derived (1-BA) cells, were grown to confluence in 60 mm tissue culture dishes (Nalge Nunc International), and pre-treated with either vehicle (DMSO) or 0.5-, 1.0- or 1.5-μM of the proteasome inhibitor MG-132 (Sigma) for 3 hr. The inhibitor was then removed, and fresh medium containing DMSO (vehicle), or CSE (various concentrations) was added, and the cells maintained for an additional 24 hr as described under “Global DNA Methylation Assay”. Control experiments, in which cells were treated with only 1.5-μM MG-132 (for 3 hr) were also performed. Subsequently, nuclear protein extracts were prepared and examined by western analysis as detailed in the previous paragraph.
60 mm tissue culture dishes
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 60-mm tissue culture dishes are a type of laboratory equipment used for cell culture applications. They provide a controlled environment for the growth and maintenance of cells in vitro. These dishes are typically made of high-quality, durable materials and are designed to support the optimal growth conditions for a variety of cell types.
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Lab products found in correlation
Mg132, by Merck Group (1 mentions)
5 azacytidine, by Merck Group (1 mentions)
Coomassie blue g 250, by Bio-Rad (1 mentions)
Facsaria 2, by BD (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
5 protocols using 60 mm tissue culture dishes
1
Proteasome Inhibitor Effects on DNA Methylation
2
Assessing Global DNA Methylation in 1-BA Cells
3
Quantification of MMP-9 and MMP-2 Levels
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U251N cells were seeded in 60-mm tissue culture dishes (Thermo Fisher Scientific, Rochester, NY, USA) at 1,500,000 cells per dish in 3 ml media and cultured for 24 h. Cells were treated in serum-deprived DMEM for 24 h. Following treatment, culture media were collected and concentrated 15-fold using 30 kDa centrifugal filters (Millipore, Cork, Ireland) following the manufacturer’s recommendations. Concentrated media were separated by SDS-PAGE using gelatin (0.1%, w/v) and acrylamide (7.5%, w/v) gels under non-reducing conditions. Gels were washed for 30 min in renaturing solution (2.5% (v/v) Triton X-100 in double-distilled water) and 30 min in developing buffer (50 mM Tris, pH 7.8; 1% (v/v) Triton X-100; 1 μM ZnCl2, 5 mM CaCl2, adjusted to pH 7.45). Gels were then incubated in fresh developing buffer at 37 °C overnight. Gels were stained with 0.5% (w/v) Coomassie Blue G250 (Bio-Rad, Richmond, CA, USA) dissolved in 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h, and then destained in 40% ethanol and 10% acetic acid diluted in double-distilled water, until clear bands appeared. Quantification of MMP-9 and MMP-2 abundance (as band area) was done in ImageJ.
4
Clonogenic Assay of KYSE Cells
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KYSE-30 or KYSE-140 cells were sorted into 1,000 cells by using BD FACSAria™ II. The cells were plated in 60-mm tissue culture dishes (Thermo Fisher Scientific). After 14 days of culturing, colonies were stained with Diff-Quik (Sysmex International Reagents, Co., Ltd., Kobe, Japan) and the number of colonies with a diameter of >3 mm was counted.
5
Ginsenoside Rg3 Induces Apoptosis
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5 × 105 H1264 and Calu-6 cells were plated in 60-mm tissue culture dishes (Thermo Scientific), grown overnight, and treated with growth medium containing 250 μM of ginsenoside Rg3 (isolated from the EA fraction) or 0.5% DMSO as a vehicle control. Cells were also treated with 1 μM doxorubicin (Sigma-Aldrich) as a positive control. At 18 or 48 h after treatment, cells were harvested and lysed with Radioimmunoprecipitation assay (RIPA) buffer supplemented with 1 μM DTT, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich), and a protease inhibitor cocktail (Roche, Mannheim, Germany). Proteins in the resultant whole cell lysates were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed for apoptosis-inducing factor (AIF), poly (ADP-ribose) polymerase (PARP), and β-Actin as a loading control using anti-AIF, anti-PARP (Cell Signaling Technology, Danvers, MA, USA), and anti-β-Actin (Thermo Scientific) antibodies, respectively.
Relative gel densities were determined by densitometric analysis using ImageJ software (NIH, Bethesda, MD, USA) and normalization to β-actin as previously described [28] (link).
Relative gel densities were determined by densitometric analysis using ImageJ software (NIH, Bethesda, MD, USA) and normalization to β-actin as previously described [28] (link).
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