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Cat. No.: 3DMMPA-20 (for 20T)

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

Description

SBS3D™ Mitochondrial Membrane Potential and Apoptosis Detection Kit uses the red fluorescent probe Mito-Tracker Red CMXRos (also known as MitoTracker Red CMXRos), which is dependent on mitochondrial membrane potential, and the green fluorescent apoptosis probe Annexin V-FITC to detect mitochondrial membrane potential and apoptosis in cell spheroids or organoids. This kit enables rapid and convenient dual staining with red and green fluorescence for assessing changes in mitochondrial membrane potential in 3D-cultured cell spheroids or organoids and staining of apoptotic cell membranes. Additionally, the kit includes Hoechst 33342 staining solution for nuclear fluorescence staining, allowing morphological observation of apoptosis through nuclear fragmentation and chromatin condensation if needed.

With just 15 minutes of staining, you can observe very bright red fluorescence differences in mitochondrial membrane potential between apoptotic and normal cells, as well as green fluorescence staining in apoptotic cell membranes under a fluorescence microscope. This kit is suitable for use with fluorescence microscopes, fluorescence plate readers, and other fluorescence detection systems.

The kit is user-friendly and yields clear results. It simultaneously detects two critical apoptosis markers: changes in mitochondrial membrane potential and phosphatidylserine externalization. Post-detection, live cells show negative green fluorescence but positive red fluorescence; apoptotic cells show positive green fluorescence and significantly reduced or negative red fluorescence, creating a sharp contrast between live and dead cells (refer to Figure 1). Detection can be performed using a fluorescence microscope, flow cytometer, or other fluorescence detection equipment.

It's important to note that red fluorescence staining may gradually diminish after apoptosis or necrosis induction, but some cells with reduced red fluorescence may not necessarily show positive green fluorescence staining. Additionally, phosphatidylserine externalization is an early event in apoptosis; early apoptotic cells may not exhibit nuclear fragmentation or chromatin condensation, typical morphological changes observed during apoptosis.

This kit detects the red fluorescence of Mito-Tracker Red CMXRos and the green fluorescence of Annexin V-FITC. It also includes Hoechst 33342, a cell membrane-permeable blue fluorescent dye with low toxicity, making it an excellent nuclear stain for live cells.

Components

• SBS3D™ Mito-Tracker Red CMXRos (100X)
• SBS3D™ Annexin V-FITC (40X)
• SBS3D™ Annexin V-FITC Binding Buffer
• SBS3D™ Hoechst 33342 Staining Solution (100X)

Features

• This kit has a wide range of applications. It can be used for 3D cell spheroids or organoids cultured by conventional methods, including ultra-low attachment cell culture 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-FITC (40X), SBS3D™ Mito-Tracker Red CMXRos (100X), and SBS3D™ Hoechst 33342 Staining Solution (100X) with SBS3D™ Annexin V-FITC 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. Except for SBS3D™ Annexin V-FITC Binding Buffer, all components should be stored protected from light.

Precautions

• Although testing has shown that SBS3D™ Annexin V-FITC (40X) can withstand up to 5 freeze-thaw cycles without significant impact on its performance, it is recommended to store it at 4ºC for 3-6 months to achieve optimal results, while avoiding repeated freeze-thaw cycles.
• 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-FITC, 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.