UA-Glo® Caspase 3/7 Assay

The UA-Glo® Caspases 3/7 Apoptosis Assay Kit is designed for quantitatively measuring the activity of Caspases 3 and 7 in cells. After adding the reagent to the target cells, the released Caspases 3/7 from cell lysis cleaves the Caspases 3/7-specific peptide luciferin-conjugated substrate in the reagent, thereby releasing luciferin. The luciferase in the reagent catalyzes the luciferin reaction to generate light, the intensity of which is proportional to the activity of Caspases 3/7. This reagent features high signal-to-noise ratio, excellent reproducibility, and superior stability. The homogeneous, ready-to-use formulation reduces experimental preparation and operational steps, minimizing errors caused by multiple pipetting. Its stable glow signal makes this product particularly suitable for high-throughput compound screening.

UA-Glo® Caspases 3/7 Apoptosis Detection Kit specifications are as follows. The number of reactions for 96-/384-well plates is calculated based on adding 100μL / 20μL of detection reagent per well.

1.    Cell Preparation

1)    Seed cells at an appropriate density in a white 96-well or 384-well cell culture plate.

2)    After incubating the cells for a specified time according to the experimental design, add the test compounds at the desired concentrations to the wells. It is recommended to include a cell control with solvent only (negative control) and a cell culture medium control with solvent only (blank control). The blank control measures the background fluorescence associated with the cell culture system and the UA-Glo® Caspase 3/7 detection reagent, and this value can be subtracted from the test results for further calculations. The cell negative control reflects the basal Caspase 3/7 activity of the test cells and serves as a reference for data interpretation.

3)    Continue culturing for an appropriate time to induce apoptosis as required by the experiment.

2.    UA-Glo® Caspases 3/7 Apoptosis Assay

1)    Remove the UA-Glo® Caspases 3/7 apoptosis detection reagent and allow it to thaw completely at room temperature (22°C–25°C), then equilibrate further to room temperature. Invert the vial to mix thoroughly. Take the required amount of reagent for the experiment, and aliquot and store the remaining reagent at -20°C or below.

2)    Remove the cell culture plate and equilibrate it to room temperature. The luciferase reaction is sensitive to temperature changes. Both the reagent and test samples must be equilibrated to room temperature, and the temperature should remain constant (±1°C) during testing.

3)    Add an equal volume of detection reagent to the cell culture medium in each well. For example, add 100μL of detection reagent to 100μL of medium.

4)    Shake the plate at medium speed for 1min, then incubate in the dark at room temperature for 10–60min.

5)    Measure the luminescence signal using a luminometer or a multifunctional plate reader. The signal typically peaks around 1hr after adding the detection reagent. The earliest reading can be taken at 30min, but no later than 2–3hr. The optimal detection window depends on the cells and experimental setup and should be optimized for each experiment.

1. It is not recommended to mix reagents from different batches.

2. Without rigorous validation, it is not advisable to alter the volume of the test reagents.

3. For research use only.