Assay Principle

The caspase 3/7 Active Apoptosis Detection Kit (Green fluorescence) is used for apoptosis detection by measuring the activity of caspase 3/7. Because caspase 3 (CPP32/ Apopain) plays a significant role in the initiation of apoptosis, it has been widely accepted as an indicator of apoptosis. caspase 3 has a specific substrate polypeptide recognition sequence, namely aspartic acid-glutamate-valine-aspartic acid (DEVD).

In this kit, caspase 3/7 Green is conjugated to a high-affinity DNA fluorescent dye using the caspase 3/7 recognition sequence(DEVD), which has cell membrane permeability and can penetrate the cell membrane to enter the cytoplasm. caspase3/7Greenreagent itself is non-fluorescent and has a charge repulsion effect with DNA. When cells undergo apoptosis, caspase3/7Green,as a substrate, is cleaved by activated caspase 3/7 and releases high-affinity DNA dyes. After binding to DNA, it generates strong fluorescence (Ex /Em: 488nm / 525 nm, flow cytometry recommended FITC channel), which is used for thedetectionofcaspase-3/7 activity and apoptosis. This kit can be used to detect the activity and apoptotic status of caspase 3/7 in cells by flowcytometry, fluorescence microplate reader and fluorescence microscope.

Materials Supplied and Storage Conditions

Reagent Preparation

Caspase 3/7 Green: Ready to use as supplied. Equilibrate to room temperature before use. The unused reagents are sub-packaged and stored at -20℃, protected from light. Avoid repeated freezing and thawing.

Z-VAD (OMe)-FMK: Ready to use as supplied, the concentration is 10 MM. Equilibrate to room temperature before use. The un used reagents are sub-packaged and stored at -20℃, protected from light. Avoid repeated freezing and thawing

Assay Procedure

Experimental Design: Select an appropriate method to induce apoptosis, and at the same time set up a control group without induction. This kit provides a certain amount of the inhibitor Z-VAD (OMe)-FMK, and the inhibitor group can be added simultaneously with induction as needed. Although Z-VAD(OMe)-FMK is a widely recognized Caspase inhibitor, in the specific experimental process, the inhibit or effect of Z-VAD (OMe)-FMK on different apoptosis inducers and apoptosis of different cells varies. Researchers need to explore the drug concentration and experimental methods by themselves according to the experimental purpose. During the development of this reagent, camptothecin and raptinal were used to induce apoptosis in hela and jurk at cells, and the addition of inhibitors showed significant inhibitory effects. In addition to Z-VAD (OMe)-FMK, other Caspase inhibitors such as Ac-DEVD-CHO can also be selected, and screening should be conducted based on the experimental purpose.

Flow cytometry analysis and microplate reader detection

• Collect cells, centrifuge at 3 00g for 5 min at 4℃, collect 1-5×10 5 cells, and wash twice with ice-cooled PBS.
• Resuspend the cells with 500 µL of PBS.
• Add 1 µL of Caspase 3/7 Green staining solution to every 500 µL of cell suspension and gently mix well.
• Incubate at room temperature in the dark for 20 to 30 min.
• Place on ice after incubation. Within 30 min after staining, the staining was detected by flow cytometry (FITC channel) or inoculated into a 96-well full black plate and detected by fluorescence microplate reader (Ex/Em: 488nm/525 nm).

Fluorescence microscopy analysis

For suspension cells

• Prepare samples according to the flow cytometry analysis steps.
• Suspend the stained cells on a glass slide and cover them with a coverslip. Analyze the cells through a fluorescence microscope as soon as possible using an appropriate filter.

For adherent cells

• Inoculate the cells onto a slide or chamber slide and culture the cells.
• Induce apoptosis of cells by the required method, and at the same time set up a control group without induction.
• Remove the culture medium and wash the cells twice with PBS.
• Prepare the working solution: Add 1 µL of Caspase 3/7 Green for every 200 µL of PBS and gently mix well.
• Add 200 µL of the prepared working solution to each well.
• Analyze the cells under a fluorescence microscope with appropriate filters