The MTT assay was used to assess FOH treatment effects on cell viability, essentially as described previously (Mosmann, 1983 (link)). Briefly, MTT was dissolved at a concentration of 1 mg/mL in phosphate-buffered saline (PBS) and sterile-filtered prior to use. Cells were cultured in 12-well plates. At the selected times following treatment, culture medium was replaced with 0.3 mL of MTT solution, and cells were further incubated for 30 minutes at 37° C. Following incubation, cells were washed with PBS, and MTT formazan was extracted with 0.3 ml 100% isopropanol. Aliquots of the isopropanol extracts were diluted 1:5 in isopropanol and absorbance was measured at 560 nm using a SpectraMax Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA). Each sample was measured in triplicate, and each experiment was repeated in 3 independent HepaRG cultures.
Spectra max plus absorbance microplate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax® Plus Absorbance Microplate Reader is a versatile laboratory instrument designed for measuring the absorbance of samples in microplate format. It is capable of performing accurate and reliable absorbance measurements across a wide range of wavelengths, making it suitable for a variety of applications in scientific research and analysis.
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Lab products found in correlation
8 protocols using spectra max plus absorbance microplate reader
1
MTT Assay for Cell Viability Evaluation
2
Cell Metabolic Activity Quantification
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Cell counting kit-8 (CCK8) assay is a colorimetric assay similar to MTT assay but has no cytotoxicity. The resulting optical density (OD) reflects cell metabolic activity level and cell viability. Wells in a 96-well plate were coated with 1% PGmatrix, followed by 30 min gelation at 37 °C, to prevent cell adhesion to plastic. hADMSCs encapsulated in 3% Hystem and 0.3% PGmatrix were plated into 3 wells for each treatment. Twenty microliters/well hADMSC-encapsulated 3% HA gel and hADMSC suspension were plated into 18 wells for each treatment. All plates were incubated at 37 °C for 1 h to allow complete gelation of PGmatrix and Hystem. CCK8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was mixed with DPBS at 1:9 ratio, 100 µL mixture/well (10 µL CCK8 solution for suspension wells) was added at 0, 3, 6, 12, 24, and 48 h after gelation and incubated for 1 h at 37 °C. Absorbance at 450 nm was measured with SpectraMax Plus Absorbance Microplate Reader (Molecular Devices, LLC., San Jose, CA, USA) right after CCK8 addition and after 1 h incubation. The final optical density (OD) of each well = OD after 1 h incubation—OD right after CCK8 addition. After measurement, the CCK8 mixture in Hystem and PGmatrix wells was replaced with DPBS. Wells containing 3% HA and suspension were discarded after measurement.
3
Competitive ELISA for Screening CtBP1 Inhibitors
4
Cell Proliferation Assay Using MTT
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Cell proliferation assay was determined by MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China). Briefly, the cells were seeded in 96-well plates at 2.5 × 104 cells/cm2 (n = 6) and cultured in a growth medium for 24 h. MTT reagent (10 μL) and serum-free basal media (90 μL) were added to each well and incubated for 4 h. The supernatant was aspirated; formazan solutions (110 μL) were added to each well to dissolve the MTT formazan. Absorbance was measured at 490 nm using the Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).
5
Glutathione Quantification in Cultured Cells
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A total glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with enzyme labelling was used to analyse GSH following the manufacturer's instructions. Briefly, the cells were seeded at 2.5 × 104 cells/cm2 in 24-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis. The supernatant (100 μL) was mixed with precipitant agents (100 μL) and centrifuged at 3500 rpm for 10 min at 25 °C. Following this, the reaction mixture was kept at room temperature for 5 min, and the absorbance values were measured at 405 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).
6
Quantifying Cellular SOD Activity
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A total superoxide dismutase (SOD) assay kit with water-soluble tetrazolium salt, WST-1 (Beyotime Biotechnology, Shanghai, China), was used to analyse the SOD enzyme activity following the manufacturer's instructions. Briefly, the cells were seeded at a density of 2.5 × 104 cells/cm2 in 96-well plates (n = 3) and treated as described previously. The cells were harvested, lysed in 1 × PBS by ultrasonic pyrolysis, and centrifuged at 2200 rpm for 5 min at 4 °C. The supernatant (100 μL) was mixed with SOD working solution (160 μL) at 4 °C. The reaction mixture was centrifuged and transferred to 96-well plates, and the absorbance values were measured at 450 nm using a Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).
7
Intracellular ROS Activity Measurement
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The intracellular ROS activity was examined by the Reactive Oxygen Species Assay Kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells were seeded at the density of 2.5 × 104 cells/cm2 in 96-well plates and cultured for 24 h (n = 3). DCFH-DA was diluted 1:1000 in serum-free medium to 10 μmol/L concentration and added to each well. The cells were incubated at 37 °C for 20 min and washed three times with a serum-free medium. The ROS activity was measured at 488 nm by Spectra Max® Plus Absorbance Microplate Reader (Molecular Devices, Sunnyvale, CA).
8
Cell Viability Assay with Rotenone
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The viability of cells was assessed with MTT assay. In brief, 1 × 104 cells were plated into 96-well plates and incubated overnight. Cells were then washed with fresh medium without serum to remove cell debris and treated with different reagents. Before treating cells with rotenone, the cells were preincubated with PA, SB203580 (p38 MAPK), SP600125 (JNK) or U0126 for 1 h, respectively. When exposed to different treatments for the indicated times duration, cells were treated with 1 mg/mL MTT for 4 h at 37°C and then with DMSO overnight. Absorbance was determined at 490 nm with SpectraMax Plus absorbance microplate reader (Molecular Devices, USA) and then normalized by scaling to the mean of control cells (defined as 100%). Each assay was performed in triplicate and repeated three times.
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