Cells were transfected as aforementioned. The C2C12 myoblasts were divided into 4 groups as follows: NC group, siMyo18b group, mock group and Myo18b group. To determine proliferation, 2×103 C2C12 myoblasts were seeded into 96-well plates and cultured in DMEM with 5% CO2 at 37°C. Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) reagent (10 µl/well) was added at 24 and 48 h, and the cells were further incubated for 3 h at 37°C. Subsequently, the absorbance at 450 nm was detected using a plate reader (Thermo LabSystems; Thermo Fisher Scientific, Inc.).
Thermo labsystems
Manufactured by Thermo Fisher Scientific
Thermo LabSystems is a line of laboratory equipment designed to support various scientific experiments and analytical procedures. It offers a range of products, including pipettes, centrifuges, incubators, and other essential lab instruments. The core function of Thermo LabSystems is to provide reliable and consistent performance to enable accurate and efficient laboratory workflows.
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5 protocols using thermo labsystems
1
C2C12 Cell Proliferation Assay
2
Caspase Activity Quantification in Cartilage
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Cartilage and synovial tissue from the three genotypes were suspended in 1 mL of lysis buffer containing 50 mM Tris HCl (pH 7.4), 0.1 mM sodium orthovanadate, 50 mM sodium fluoride, 150 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 5 µg/mL pepstatin A. Mixtures were homogenized and microcentrifuged at 14,000 rpm for 15 min at 4 °C. The protein content of the supernatant was determined using a detergent compatible-protein assay (Bio-Rad, Hercules, CA, USA). A total of 100 µL of diluted (10 μg/mL) extract was mixed with 100 µL Caspase-Glo® assay kit (Promega, Madison, WI, USA) in a 96-well plate. Briefly, wells were then gently mixed with a plate shaker at 300–500 rpm for 30 s [51 (link)] and was incubated at room temperature for 2 h. The luminescence of each sample was measured in a plate-reading luminometer (Thermo Labsystems, Thermo Fisher Scientific) with parameters of 1 min lag time and 0.5 s/well acquisition time. All experiments were performed in triplicate.
3
Cell Viability Assay for Breast Cancer
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Following transfection, the breast cancer cells (2×104/well) in each group were plated in 96-well plates and incubated for 24 h at 37°C. Subsequently, 10 µl of CCK-8 solution (Dojindo Molecular Technologies, Inc.) were added to each well to incubate the cells for 1 h at 37°C. The absorbance at a wavelength of 450 nm was measured using a microtiter plate (Thermo LabSystems; Thermo Fisher Scientific, Inc.).
4
Cell Proliferation Assay with Y27632
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Cells were plated into 96-well plates in triplicate at ~10,000 cells/well and subsequently cultured with or without 10 µM Y27632 at 37°C and 5% CO2 for 24 h. Cells were analyzed using CCK-8 assay according to the manufacturer's protocol (Beyotime Institute of Biotechnology). In total, 10 µl CCK-8 were added per well and incubated for 2 h at 37°C. The absorbance value at a wavelength of 450 nm was measured for each well using an enzyme-linked immunosorbent assay reader (Thermo Lab Systems; Thermo Fisher Scientific, Inc.).
5
Cytotoxicity of Soybean Extracts on A375 and 451Lu Cells
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The effect of NBSE and BSE soybean extracts on the viability of A375 and 451Lu cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay [31] (link). The cells (70% confluent) were treated with NBSE and BSE (0–2.2 mg/mL for A375, and 0–1.6 mg/mL for 451Lu) in complete culture medium in 24-well microtiter plates for 24 h at 37°C and 5% CO2 in a humidified chamber. Twenty-four hours post-treatment, 300 µL of MTT reagent (0.5 mg/mL final concentration in medium) was added to each well and incubated for 2 h. The MTT solution was removed from the wells by aspiration and the formazan crystals were dissolved in DMSO (300 µL). Absorbance was recorded on a microplate reader (ThermoLabsystems, Fisher) at 540 nm wavelength. Vehicle (DMSO)-treated cells were regarded as 100% viable.
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