The cytotoxicity of ATRA and/or fulvestrant was evaluated using a WST-8 cell counting kit according to the manufacturer’s instructions (Sigma‒Aldrich, Germany). Briefly, 104 cells per well were seeded in 96-well plates and incubated for 48 h in DMEM, 4.5 g/L (10% FBS, 1% PS). The medium was then removed and replaced with ATRA (1, 5, 10, 20 µM) and/or fulvestrant (0.1, 0.5, 1, 2, 5, 10, 20 µM). After 48 h, 10 µl of tetrazolium salt was added to each well. This assay uses tetrazolium salt, which is converted to the fluorescent product formazan by metabolically active cells. Fluorescence was monitored at 450 nm by a Multiskan Go ELISA reader.
Wst 8 cell counting kit
Manufactured by Merck Group
Sourced in Germany
The WST-8 cell counting kit is a colorimetric assay used to measure the number of viable cells in a sample. The kit utilizes a water-soluble tetrazolium salt that is reduced by cellular dehydrogenases to produce a colored formazan dye, which is directly proportional to the number of living cells. This method provides a simple, rapid, and quantitative assessment of cell viability and proliferation.
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5 protocols using wst 8 cell counting kit
1
Cytotoxicity Assay of ATRA and Fulvestrant
2
Cell Proliferation Assay using WST-8
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Cell proliferation was determined using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) cell counting kit (Sigma-Aldrich, Germany). Briefly, 105 cells were plated in each well of a 96-well plate in quadruplicate, allowed to grow for 48 hours, and then treated with different inhibitors for another 48 hours. Tetrazolium salt was then added to each well, and the formazan formation was assessed using an ELISA reader at 450 nm. Cell proliferation was also examined using trypan blue (Sigma-Aldrich, Germany).
3
Hyperthermic Exposure and Cell Viability
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Cells were seeded in 96 well-plates with 100 μL medium and incubated overnight before hyperthermic exposure. The next day, the medium was refreshed and cells were exposed to 42°C or 43°C for 30 minutes, or for 1 hour in a 5% CO2 incubator. Cells maintained at 37°C were set as control. At the end of exposure, cells were returned back to 37°C for recovery. Cell viability levels were determined at 4, 24, 48, and 72 hours post exposure, by using the WST-8 cell counting kit; according to the manufacturer’s protocol (Sigma-Aldrich, Germany). This assay uses tetrazolium salt which is converted to a fluorescent product formazan by metabolically active cells. Fluorescence was monitored at 450 nm by ELISA reader Multiskan Go. Cell viability was expressed as a percentage of control cells. Five replicates (n = 5) of each experimental condition were performed.
4
Cytotoxicity Evaluation of DKK1
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The cytotoxicity of DKK1 was evaluated using a WST-8 cell counting kit according to the manufacturer’s instructions (Sigma-Aldrich, Germany). Briefly, 104 cells per well were seeded in 96-well plates and incubated for 48 hours in DMEM/F12 (10% FBS). The medium was then removed and replaced with recombinant DKK1 solutions (0.10, 100, 150, 200 ng/ml). After 24, 48 and 72 hours, 10 μl of tetrazolium salt was added to each well, and the formazan formation was assessed using an ELISA reader Multiskan Go at 450 nm.
5
Evaluating Gingival Fibroblast Viability
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Following culture with a human gingival fibroblast cell line (p10-13) in a 24-well plate (1×10 4 /well) for 24 h, the sample was placed in cell culture inserts so that it would account for 10% of the entire culture medium (1,500 μL/ well). The sample was cultured at 37°C under 5% CO 2 for 24 h. Next, the optical density (OD) measured at 450 nm (n=5) using a WST-8 cell counting kit (Sigma-Aldrich Japan, Tokyo, Japan) 20) . For apoptosis measurements, the cells (1×10 4 /well) were exposed to the specimens in 24-well plates for 24 h. Following exposure, the cells were trypsinized, centrifuged and resuspended in 50 μL fetal bovine serum (FBS) containing 10 μg/mL Hoechst 33342 (Thermo Fisher Scientific, Tokyo, Japan) and 10 μg/mL propidium iodide (Sigma, St. Louis, MO, USA). After incubation for 30 min in dark at room temperature, the cells were spread onto microscope slides. A minimum of 300 cells were counted using a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan) with an excitation filter of 340-380 nm, and the cells were classified as apoptotic, necrotic, or viable.
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