To quantify GFP expression, cells were plated in clear flat-bottomed 96-well plates (Nunc) and imaged every hour using the IncuCyte S3 live cell imaging system (Essen BioScience). Five fields of view were taken per well at 10× magnification, and GFP expression was determined using the total integrated intensity metric included in the IncuCyte S3 software (Essen BioScience). To analyse images generated on the IncyCyte S3, a collection of representative images is first taken to set fluorescence and cellular thresholds, which allows for the removal of background fluorescence, and selection of cell boundaries (‘objects’) by creating ‘masks’. Following this, the total integrated intensity metric can be accurately calculated by the software, which takes the total sum of objects’ fluorescent intensity in the image, expressed as green count units (GCU) µm−2.
Clear flat bottomed 96 well plates
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Clear flat-bottomed 96-well plates are laboratory equipment designed for various scientific applications. These plates provide a standard format with a flat bottom and 96 individual wells, enabling efficient sample handling and processing.
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3 protocols using clear flat bottomed 96 well plates
1
IncuCyte S3 Quantification of GFP
2
MTT Assay for Cytotoxicity Evaluation
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The MTT assay was performed to monitor the cytotoxic effects of test compounds in the RGA cell lines. Briefly, clear flat-bottomed 96-well plates (Nunc, Roskilde, Denmark) were seeded with 4 x 10 5 cells/ml of the appropriate cell line. After 24 h
convert the soluble yellow MTT into insoluble purple formazan by the action of mitochondrial succinate dehydrogenase. Following incubation, supernatant was discarded and 50 µl of MTT solution/well (5mg/ml stock in PBS diluted in 1:2.5 in assay media) was added and cells were incubated for a further 3 h. The supernatant was removed and 200 µl of DMSO was added to each well and incubated for 10 min with agitation at 37 °C to dissolve the formazan crystals. Optical density was measured using a Sunrise spectrophotometer at 570 nm with a reference filter at 630 nm (TECAN, Switzerland). Samples were analysed in triplicate wells and in three independent experiments. Viability was calculated as a percentage absorbance of the sample when compared with the absorbance of the solvent control (Fig. 1).
convert the soluble yellow MTT into insoluble purple formazan by the action of mitochondrial succinate dehydrogenase. Following incubation, supernatant was discarded and 50 µl of MTT solution/well (5mg/ml stock in PBS diluted in 1:2.5 in assay media) was added and cells were incubated for a further 3 h. The supernatant was removed and 200 µl of DMSO was added to each well and incubated for 10 min with agitation at 37 °C to dissolve the formazan crystals. Optical density was measured using a Sunrise spectrophotometer at 570 nm with a reference filter at 630 nm (TECAN, Switzerland). Samples were analysed in triplicate wells and in three independent experiments. Viability was calculated as a percentage absorbance of the sample when compared with the absorbance of the solvent control (Fig. 1).
3
Cytotoxicity Evaluation of Steviol Compounds
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The MTT assay was performed to monitor the toxic effects of test compounds in the RGA cell lines. Briefly, clear flat-bottomed 96-well plates (Nunc, Roskilde, Denmark) were seeded with 4 x 10 5 cells/ml of the appropriate cell line. After 24 h stevioside and steviol (500, 1,000, 5,000, 10,000 and 25,000 ng/ml) and rebaudioside A (5,000, 10,000, 25,000, 50,000 and 100,000 ng/ml) were added to the cells at a final DMSO concentration of 0.1%. Test compounds were then incubated for a further 48 h. Viable cells convert the soluble yellow MTT into insoluble purple formazan by the action of mitochondrial succinate dehydrogenase.
Following incubation, supernatant was discarded and 50 µl of MTT solution/well (5mg/ml stock in PBS diluted in 1:2.5 in assay media) was added and cells were incubated for a further 3 h. The supernatant was removed and 200 µl of DMSO was added to each well and incubated for a further 10 min with agitation at 37°C to dissolve the formazan crystals.
Optical density was measured using a Sunrise spectrophotometer at 570 nm with a reference filter at 630 nm (TECAN, Switzerland). Samples were analysed in triplicate wells and in three independent experiments. Viability was calculated as a percentage absorbance of the sample when compared with the absorbance of the DMSO solvent control.
Following incubation, supernatant was discarded and 50 µl of MTT solution/well (5mg/ml stock in PBS diluted in 1:2.5 in assay media) was added and cells were incubated for a further 3 h. The supernatant was removed and 200 µl of DMSO was added to each well and incubated for a further 10 min with agitation at 37°C to dissolve the formazan crystals.
Optical density was measured using a Sunrise spectrophotometer at 570 nm with a reference filter at 630 nm (TECAN, Switzerland). Samples were analysed in triplicate wells and in three independent experiments. Viability was calculated as a percentage absorbance of the sample when compared with the absorbance of the DMSO solvent control.
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