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Cat. No.: 3DC3AA-20 (for 20T)

Cat. No.: 3DC3AA-50 (for 50T)

Description

SBS3D™ Caspase-3 Activity and Apoptosis Detection Kit is based on the novel cell-permeable Caspase-3/7 green fluorescent substrate GreenCell™ Caspase-3 Substrate, combined with the red fluorescent apoptosis probe Annexin V-mCherry. This kit is designed to detect Caspase-3 activity and phosphatidylserine externalization on the cell membrane during apoptosis in cell spheroids or organoids, using dual red and green fluorescence staining.

This kit enables rapid and convenient detection of Caspase-3 activity and apoptosis in 3D-cultured cell spheroids or organoids, with red fluorescence staining of apoptotic and necrotic cell membranes. It is suitable for real-time monitoring of Caspase-3 activity and apoptosis in live cells. With just 15 minutes of staining, apoptotic cells can be observed under a fluorescence microscope with bright red fluorescence in the cell membrane and green fluorescence in the nucleus, while necrotic cells show only red fluorescence in the cell membrane. This kit is compatible with fluorescence microscopes, fluorescence plate readers, and other fluorescence detection systems.

The kit detects green fluorescence from GreenCell™-DNA complexes and red fluorescence from Annexin V-mCherry. The maximum excitation wavelength for GreenCell™-DNA is 500nm, with a maximum emission wavelength of 530nm. For Annexin V-mCherry, the maximum excitation wavelength is 587nm, and the maximum emission wavelength is 610nm.

Normal cells are not stained by GreenCell™ and Annexin V-mCherry. Apoptotic cells with high Caspase-3 activity exhibit bright green fluorescence in the nucleus and red fluorescence staining by Annexin V-mCherry.

Components

• SBS3D™ Annexin V-mCherry (40X)
• SBS3D™ GreenCell™ Caspase-3 Substrate (100X)
• SBS3D™ Annexin V-mCherry Binding Buffer

Features

• This kit allows real-time monitoring of Caspase-3/7 activity in cells. Traditional Caspase activity detection kits typically require cell or tissue sample lysis followed by colorimetric or fluorescence detection. Western blotting for detecting Caspase and its cleaved protein expression only reflects enzyme activity to a certain extent. The Fluorochrome-Labeled Inhibitors of Caspases (FLICA) technique, while able to enter live cells to detect Caspase activity, uses irreversible Caspase inhibitors, making real-time monitoring in live cells impossible. In contrast, the fluorescent substrate GreenCell™ Caspase-3 provided in this kit does not significantly inhibit cell viability, Caspase expression, or the apoptosis process, facilitating real-time monitoring of apoptosis.
• This kit is highly functional. While detecting Caspase-3/7 activity, it also allows real-time observation of nuclear morphological changes during apoptosis and detection of phosphatidylserine (PS) externalization, a classic event in apoptosis. Apoptotic cells may be at different stages and undergo various events; thus, it is crucial to independently track these events in real-time to better understand their relationships. This kit enables the study of these temporal and spatial relationships through fluorescence microscopy.
• The kit is versatile. It can be used for 3D cell spheroids or organoids cultured by conventional methods, including ultra-low attachment plates, Matrix-Gel or Matrigel-coated plates, agarose-coated plates, and hanging drop culture plates.
• The kit is user-friendly, with the entire detection process taking only about 15-30 minutes. After inducing apoptosis or necrosis in 3D cell spheroids, prepare the working solution by mixing SBS3D™ Annexin V-mCherry (40X) and SBS3D™ GreenCell™ Caspase-3 Substrate (100X) with SBS3D™ Annexin V-mCherry Binding Buffer in the appropriate ratios. Incubate in the dark for 15 minutes, and then proceed with fluorescence microscopy imaging and analysis.

Storage

Store at -20ºC for one year. SBS3D™ Annexin V-mCherry (40X) and SBS3D™ GreenCell™ Caspase-3 Substrate (100X) should be stored protected from light.

Precautions

• The GreenCell™ Caspase-3 Substrate is not very stable and should be protected from repeated freeze-thaw cycles (preferably no more than three times).
• Cell spheroids can easily deform or disperse under external force; therefore, processes like PBS washing and media changes should be performed gently to avoid disrupting or dispersing 3D cell spheroids.
• Different types of cell spheroids may have varying tolerance to apoptosis or necrosis inducers. After inducing apoptosis or necrosis in 3D cell spheroids, their morphology may change. It is advisable to observe the cell spheroid morphology under a microscope before staining and consider selecting relatively intact spheroids for staining analysis.
• Bacterial or fungal contamination can severely affect detection results.
• It is advisable to perform detection as soon as possible after staining, as prolonged time may increase the number of apoptotic or necrotic cells.
• Fluorescent substances are prone to quenching; therefore, minimize observation time during fluorescence observation and protect from light during handling and storage.
• If trypsin was used during cell collection, ensure the removal of residual trypsin. Residual trypsin can digest and degrade SBS3D™ Annexin V-mCherry, leading to staining failure.
• This product is intended for scientific research use by professionals only. It is not suitable for clinical diagnosis or treatment, food or pharmaceutical use, and should not be stored in ordinary residential areas.
• For your safety and health, please wear a lab coat and disposable gloves when handling.