UA-Glo® Caspase 3/7 3D Assay

The UA-Glo® Caspase 3/7 3D Cell Apoptosis Assay Kit is used for the quantitative detection of Caspase 3 and Caspase 7 activity in 3D cell models. After the reagent is added to the 3D cells being tested, the intracellular Caspases 3/7 cleave the Caspase 3/7–specific fluorescently labeled peptide substrate within the reagent, releasing the fluorophore. The luciferase in the reagent then catalyzes a luminescent reaction, generating light; the intensity of this light is directly proportional to the activity of Caspases 3/7. The UA-Glo® Caspase 3/7 3D Cell Apoptosis Assay contains an optimized cell lysis reagent that enhances lysis of 3D cells, offering high signal-to-noise ratio, good reproducibility, and excellent stability. Its ready-to-use homogeneous formulation reduces experimental preparation and handling steps, minimizing errors caused by multiple pipetting operations. Additionally, its stable glow-type signal (detection signal half-life > 3 hours) makes this product particularly suitable for high-throughput compound screening.

UA-Glo® The specifications for the Caspases 3/7 3D Cell Apoptosis Assay Kit are as follows. The number of reactions that can be detected in a 96-/384-well plate is calculated based on adding 100 μL / 20 μL of assay reagent per well.

1.Cell Preparation

1)Construct the relevant 3D cell model according to experimental requirements, such as a 3D cell spheroid model.

2)Add the compound to be tested at an appropriate concentration to the wells of the cell plate based on the experimental design. It is recommended to set up a cell control with only the solvent carrier (negative control) and a medium control with only the solvent carrier (blank control). The blank control measures the background fluorescence associated with the cell culture system and the UA-Glo® Caspase 3/7 3D apoptosis assay reagent, while the cell negative control reflects the basal Caspase 3/7 activity of the cells being tested and can serve as a reference for interpreting experimental results. Detection correction values can be used by subtracting the blank control or cell negative control from the measured values before performing further calculations.

3)Continue culturing for an appropriate time to induce cell apoptosis according to experimental needs.

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

1)Remove the UA-Glo® Caspases 3/7 3D Cell Apoptosis Assay Reagent from storage. After completely thawing at room temperature (22°C–25°C), equilibrate further to room temperature and mix thoroughly by inverting. Take out the assay reagent needed for the experiment; aliquot the remaining reagent as required and store it at –20°C or below.

2)Take out the experimental cell plate and equilibrate it to room temperature. The luciferase reaction is sensitive to temperature changes. Both the reagents and the test samples must be equilibrated to room temperature, and the temperature during the assay should be kept constant (±1°C).

3)The UA-Glo® 3/7 3D Cell Apoptosis Assay should be performed in a bottom-transparent or opaque white assay plate (an opaque white plate is preferred). If the experimental cell plate is fully transparent or black, transfer the 3D cell model to a bottom-transparent or opaque white assay plate before performing the assay.

4)Add the detection reagent to the experimental cell plate in an equal volume to the culture medium in each well, for example, add 100 μL of detection reagent to 100 μL of culture medium.

5)Shake the plate at room temperature for 1–5 minutes, then incubate in the dark at room temperature for 30–60 minutes (for dense 3D cells, you can appropriately extend the shaking time or incubate on a shaker at 37°C, and then equilibrate to room temperature before detection).

6)On a luminometer or multifunctional plate reader, select the "LUM" mode to read the luminescence signal. The detection signal usually reaches its maximum about 30–60 minutes after adding the detection reagent. The latest detection time should not exceed 3 hours. The optimal detection time depends on the cells and experimental setup and needs to be optimized for each experiment.

3.UA-Glo® Caspase 3/7 apoptosis assay for 3D cells (cells embedded in Matrigel)

1)After treating the 3D cells with the test molecule (compound or macromolecule), carefully aspirate the supernatant from the Matrigel-embedded cells using a pipette or an automated liquid handling workstation.

2)Add an equal volume of Corning Cell Recovery Solution (or another equivalent reagent) pre-cooled to 2°C–8°C to each well. For a 96-well plate, the typical volume is 50 μL. Seal the plate with a sealing film.

3)Place the cell plate on ice and incubate in an ice bath for 1 hour to dissociate the Matrigel polymer.

4)During the aforementioned cell ice bath, remove UA-Glo®. Caspase 3/7 3D Cell Apoptosis Assay Reagent: After completely thawing at room temperature (22°C–25°C), invert the tube several times to mix thoroughly. Dispense the assay reagent required for the experiment and keep it on ice; aliquot the remaining reagent as needed and store it at −20°C or lower.

5)Add an equal volume of Caspases 3/7 3D cell apoptosis detection reagent to each well. Shake the plate on a plate shaker for 1 minute (~500 rpm), and incubate in the dark at room temperature for at least 30 minutes.

7)On a luminometer or multifunctional plate reader, select the "LUM" mode to read the luminescence signal. The detected signal typically reaches its maximum approximately 60 minutes after adding the detection reagent. The latest detection time should not exceed 3 hours. The optimal detection time depends on the cells and experimental setup and needs to be optimized for each specific experiment.

It is not recommended to mix different batches.