The live cell assay kit contains SuperView 488 Caspase-3 Substrate and Ac-DEVD-CHO Caspase-3/7 Inhibitor. SuperView 488 Caspase-3 Substrate provides an effective tool for detecting cell apoptosis based on Caspase-3/7 activity, and is suitable for fluorescence microscopy observation and flow cytometry analysis.

Compared with other Caspase fluorescent substrates or fluorescent inhibitors based on Fluorescent Labeled Inhibitor of Caspases (FLICA) technology, the substrate provided in the kit can detect Caspase-3/7 activity without inhibiting the apoptotic process of intact cells. The substrate consists of a fluorescent DNA dye conjugated with the Caspase-3/7 DEVD recognition sequence. Initially non-fluorescent, the substrate can penetrate the cell membrane and enter the cytoplasm. In apoptotic cells, Caspase-3/7 cleaves the substrate to release the high-affinity DNA dye, which migrates to the nucleus, labels DNA, and emits bright green fluorescence. The substrate is bifunctional, capable of both detecting Caspase-3/7 activity and visualizing the morphological changes of the nucleus during cell apoptosis. Cells stained with SuperView can be fixed with formaldehyde and are compatible with subsequent immunostaining experiments.

Spectral Properties (SuperView)

Excitation/Emission = 500/530 nm (when bound to DNA)

Product Components

BSAK-053 A |  SuperView 488 Caspase-3 Substrate, 0.2 mM in DMSO  | 125 µL |  500 µL

BSAK-053 B |  SuperView 488 Caspase-3 Substrate, 0.2 mM in DMSO  | 20 µL |  100 µL

Shipping and Storage Conditions

Shipping and Storage Conditions

Precautions

• Cells can be co-stained with Hoechst 33342 dye (recommended concentration: 1 μM) to produce blue fluorescent staining of the nucleus (Excitation/Emission = 346/460 nm).
• SuperView 488 dye can be fixed with formaldehyde but is incompatible with methanol fixation
• SuperView 488-stained cells fixed with formaldehyde can be treated with 0.1% Triton X-100 for subsequent staining, but this treatment may reduce the staining intensity
• SuperView 488-stained cells fixed with formaldehyde can be treated with 0.1% Triton X-100 for subsequent staining, but this treatment may reduce the staining intensity
• This product is for research use only!

Usage Instructions

Experimental Optimization: The experimental procedures provided below are designed based on the endpoint assay method. SuperView 488 Substrate can be used for the study of long-term cell incubation processes. Cell density, substrate concentration, and inhibitor concentration may need to be optimized; the recommended substrate concentration is between 1-10 μM. Cells can be incubated with the substrate in culture medium, PBS, or other buffers. For adherent cells, it is recommended to replace with fresh medium containing the substrate to avoid uneven background. After substrate incubation, operations such as medium change or cell washing can be performed as needed.

Control Setup

It is recommended to set up the following controls:

• Negative Control: Cells without apoptosis induction
• Positive Control: Cells with apoptosis induction
• Positive Control: Cells with apoptosis induction

Inhibitor Control

The Caspase-3/7 Inhibitor Ac-DEVD-CHO provided in the kit can be used to confirm whether the fluorescent signal of SuperView 488 is dependent on Caspase-3/7 activity. For the inhibitor control, the final concentration of the inhibitor should be at least twice the substrate concentration (e.g., when using 5 μM SuperView 488 Substrate, the concentration of Ac-DEVD-CHO should be 10 μM). Incubate Ac-DEVD-CHO at room temperature for 15-30 minutes before adding the substrate, and keep the inhibitor in the incubation solution after adding the substrate. Ac-DEVD-CHO is a reversible competitive inhibitor. For effective Caspase-3/7 inhibition in certain cell types, it may be necessary to use an irreversible inhibitor (such as Z-DEVD-FMK) or add the inhibitor before or during apoptosis induction.

Flow Cytometry Analysis

Select an appropriate method to induce cell apoptosis, and use untreated cell samples as controls.4.2 For adherent cells, digest the cells with trypsin or other methods before the experiment.4.3 Resuspend the cells with cell culture medium or an appropriate buffer to a cell density of 10^6 cells/mL.4.4 Add 200 μL of cell suspension to a flow cytometry tube.4.5 For inhibitor control samples, treat the cells with Ac-DEVD-CHO as described above.4.6 Add 5 μL of 0.2 mM Substrate to 200 μL of cell suspension and mix immediately to a final substrate concentration of 5 μM. The optimal substrate concentration may vary for different cell types and needs further optimization.4.7 Incubate at room temperature in the dark for 15-30 minutes.4.8 Add 300 μL of cell culture medium or PBS, analyze using a flow cytometer, and select the channel for detecting green fluorescence (Excitation/Emission: 485/515 nm).

Fluorescence Microscopy Observation

Select an appropriate method to induce cell apoptosis, and use untreated cell samples as controls.5.2 For inhibitor control samples, treat the cells with Ac-DEVD-CHO as described above.5.3 Replace the medium of the cells with fresh culture medium or PBS containing 5 μM Substrate (see Experimental Optimization). For the inhibitor control group, incubate the inhibitor together with the substrate.5.4 Incubate at room temperature for 30 minutes or longer.5.5 Wash the cells with PBS or other buffers, observe the cells under a fluorescence microscope, and use a filter suitable for green fluorescence (Excitation/Emission: 485/515 nm).

Fluorescence Microplate Reader Detection

Culture adherent cells in a black 96-well plate; for suspension cells, adjust the density to 10^6 cells/mL and add 0.2 mL of cell suspension to each well.6.2 Select an appropriate method to induce cell apoptosis, and use untreated cell samples as controls. (Note: Cells can be treated in tubes or flasks first and then transferred to the 96-well detection plate.)6.3 For inhibitor control samples, treat the cells with Ac-DEVD-CHO as described above.6.4 For suspension cells, directly add the substrate and mix well. For adherent cells, replace the medium with fresh culture medium or PBS containing 5 μM Substrate (see Experimental Optimization). For the inhibitor control group, incubate the inhibitor together with the substrate.6.5 Cells can be observed directly in the medium containing the substrate.6.6 For suspension cells, gently shake to resuspend the cells. Set the fluorescence microplate reader to an excitation wavelength of 488 nm and an emission wavelength of 520 nm. It is recommended to use the bottom reading mode for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings.