Kinetic of cytotoxicity and proliferation assay
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Description
A robust cell-based assay to determine kinetic of compound potency and efficacy based on live-cell imaging.
Features of the assay
- 11 serial dilutions of tested compound in three replicates.
- Number of live/dead cells is measured at 24, 48 and 72 hours of treatment.
- IC50, relative growth and percentage of growth inhibition (GI50, TGI, LC50) calculations.
- Available as high-throughput screening.
- Accurate
Detailed description
Numerous methods that rely on proportionality between cell number and a cell's functional activity or a cellular marker are currently used to estimate cell number in cell viability assays. If a test compound affects the same functional activity or marker levels used to estimate cell number, the outcome of these methodologies will not be representative exclusively of cell number but also of the changes in that biomarker. This limitation is of extreme relevance for compounds which affect cell cycle progression or metabolic activity, as many anticancer agents. Furthermore, these methodologies do not discriminate reduced proliferation from increased cell death. Finally, they do not allow to monitor cell number more then one time in each well, which prevents to check the variability in the amount of cells in each sample immediately before compound treatment.
To overcome these limitations, we validated a cytotoxicity and proliferation assay that directly count nuclei, which are stained with a far-red fluorescent dye (Spirochrome SiR-Hoechst), and dead cells, stained with a green fluorescent dye (Promega CellTox) (Figure 1). Both dyes are non-toxic and can be excited multiple times, enabling to monitor cell number over time with multiple readings. A representative experiment is shown in next figures. RH4 cells were treated with serial dilutions of a cytotoxic drug and number of cells was measured at 24, 48 and 72 hours of treatment. Fluorescence images after 72 hours for a representative well of each condition, red for nuclei and green for dead cells, are shown in Figure 2. Relative growth and percentage of cell death over time are also represented to determine the kinetic of efficacy (Figures 4 and 5).
Features of the assay
- 11 serial dilutions of tested compound in three replicates.
- Number of live/dead cells is measured at 24, 48 and 72 hours of treatment.
- IC50, relative growth and percentage of growth inhibition (GI50, TGI, LC50) calculations.
- Available as high-throughput screening.
- Accurate
- Number of cells is estimated by counting nuclei.
- Fraction of dead cells is excluded by live cell counting.
- Multiple reads of the same plate allows well-to-well normalized data analysis.
- Cytostatic/cytotoxic compound discrimination.
- Phase contrast images available on request to evaluate cell morphology.
Detailed description
Numerous methods that rely on proportionality between cell number and a cell's functional activity or a cellular marker are currently used to estimate cell number in cell viability assays. If a test compound affects the same functional activity or marker levels used to estimate cell number, the outcome of these methodologies will not be representative exclusively of cell number but also of the changes in that biomarker. This limitation is of extreme relevance for compounds which affect cell cycle progression or metabolic activity, as many anticancer agents. Furthermore, these methodologies do not discriminate reduced proliferation from increased cell death. Finally, they do not allow to monitor cell number more then one time in each well, which prevents to check the variability in the amount of cells in each sample immediately before compound treatment.
To overcome these limitations, we validated a cytotoxicity and proliferation assay that directly count nuclei, which are stained with a far-red fluorescent dye (Spirochrome SiR-Hoechst), and dead cells, stained with a green fluorescent dye (Promega CellTox) (Figure 1). Both dyes are non-toxic and can be excited multiple times, enabling to monitor cell number over time with multiple readings. A representative experiment is shown in next figures. RH4 cells were treated with serial dilutions of a cytotoxic drug and number of cells was measured at 24, 48 and 72 hours of treatment. Fluorescence images after 72 hours for a representative well of each condition, red for nuclei and green for dead cells, are shown in Figure 2. Relative growth and percentage of cell death over time are also represented to determine the kinetic of efficacy (Figures 4 and 5).
