實驗概要
PrestoBlue? Cell Viability Reagent is a ready‐to‐use reagent for rapidly evaluating the viability and proliferation of a wide range of cell types. PrestoBlue? reagent is quickly reduced by metabolically active cells, providing a quantitative measure of viability and cytotoxicity in as little as 10 minutes. Protect reagent from long term exposure to light and avoid contamination, as this can reduce sensitivity.
實驗步驟
1. PrestoBlue? Cell Viability Reagent is supplied as a 10X solution. Add PrestoBlue? Reagent directly to cells in culture medium. See below for example volumes:
Format | Volume of cells media | Volume of PrestoBlue? Reagent |
Cuvette | 900 μL | 100 μL |
96-well plate | 90 μL | 10 μL |
384-well plate | 36 μL | 4 μL |
Note: Correct for background fluorescence by including control wells containing only cell culture media (no cells) on each plate.
2. Incubate ≥10 minutes at 37o C. Longer incubation times will increase sensitivity of detection. As this is a live cell assay, readings may be taken at multiple time points to determine optimal performance in your lab.
Format | Recommended Incubation Time |
Bottom-read fluorescence | 10 minutes – 2 hours |
Top-read fluorescence | 30 minutes – 2 hours |
Absorbance | 20 minutes – 2 hours |
Room temperature incubation | 10 minutes – 2 hours |
Low cell number (< 5,000 cells/100 μL) | 20 minutes – 24 hours |
3. Read fluorescence or absorbance. Fluorescence is more sensitive than absorbance and is the preferred detection method.
Format | Excitation | Emission |
General | 540–570 nm | 580–610 nm |
Fluorescence | 560 nm (10 nm bandwidth) | 590 nm (10 nm bandwidth) |
Fluorescence (Filter) | 535 nm (25 nm bandwidth) | 615 nm (10 nm bandwidth) |
Absorbance | 570 nm | 600 nm (reference wavelength for |
4. Calculate and plot the results. Higher fluorescence or absorbance values correlate to greater total metabolic activity.
Format | Instructions |
Fluorescence | l a. Optional: Average the fluorescence values of the no-cell control wells and l b. Plot fluorescence vs. experimental condition (cell number, compound |
Absorbance | l a. Normalize the 570 nm values to the 600 nm values for the experimental wells. l b. Plot the normalized 570 nm absorbance values vs. experimental condition (cell |