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Cat. No.: 3DAVP-20 (for 20T)

Cat. No.: 3DAVP-50 (for 50T)

Cat. No.: 3DAVP-100 (for 100T)

Description

SBS3D™ Annexin V-PE Apoptosis Detection Kit uses phycoerythrin (PE)-labeled recombinant human Annexin V to detect phosphatidylserine on the cell membrane surface during apoptosis of cell spheroids or organoids. This kit allows for rapid and convenient staining of apoptotic and necrotic cells in 3D cultured cell spheroids or organoids. With just 15 minutes of staining, bright red fluorescence can be observed on the cell membranes of apoptotic and necrotic cells under a fluorescence microscope. This kit is suitable for use with fluorescence microscopes, fluorescence microplate readers, and other fluorescence detection systems.

Annexins are a class of calcium-dependent phospholipid-binding proteins widely distributed in the cytoplasm of eukaryotic cells, involved in intracellular signal transduction. However, only Annexin V has been reported to regulate the activity of certain PKC proteins.

Annexin V selectively binds to phosphatidylserine (PS). Phosphatidylserine is mainly located on the inner side of the cell membrane, adjacent to the cytoplasm. During the early stages of apoptosis, various types of cells flip phosphatidylserine to the outer surface of the cell membrane. Exposed phosphatidylserine on the cell surface promotes coagulation and inflammatory responses. By binding to the exposed phosphatidylserine, Annexin V blocks its pro-coagulant and pro-inflammatory activities. For necrotic cells with lost membrane integrity, Annexin V-PE can enter the cytoplasm and bind to phosphatidylserine on the inner side of the cell membrane, causing necrotic cells to exhibit red fluorescence.

Annexin V labeled with the red fluorescent probe PE (Annexin V-PE) can be used to easily and directly detect the externalization of phosphatidylserine, a key feature of apoptosis, with fluorescence microscopes or other fluorescence detection devices.

This kit detects red fluorescence, making it especially suitable for detecting apoptosis in cells already expressing green fluorescent proteins (e.g., GFP). PE can be excited by 495nm, 545nm, or 564nm light, emitting fluorescence with a peak at 575nm.

Normal cells will not be stained by Annexin V-PE, while apoptotic and necrotic cells will be stained. To further distinguish between apoptotic and necrotic cells, other 3D cell apoptosis detection kits (e.g., SBS3D™ Annexin V-FITC Apoptosis Detection Kit) can be used.

Traditional cell culture is predominantly conducted in a two-dimensional (2D) format. However, cells grown in 2D culture differ significantly from those in vivo in terms of growth patterns, morphology, differentiation, and function. These differences, stemming from the lack of proper cell architecture and tissue morphology, can undermine the reliability of experimental results. In contrast, three-dimensional (3D) cell culture better simulates the microenvironment in which cells exist in vivo, more accurately representing in vivo tissues and reflecting interactions between cells, and between cells and the matrix. The response of cells to exogenous and endogenous stimuli in 3D culture closely mimics their in vivo reactions, making 3D cell culture a more valuable and reliable in vitro experimental model that yields results more consistent with in vivo experiments.

3D tumor cell models are increasingly being used to understand disease mechanisms and in drug development. While 2D cultured tumor cells can uniformly receive nutrients and oxygen from one side, cells within 3D cultured tumor spheroids have less access to these resources, forming natural gradients of nutrients and oxygen. This better simulates the in vivo microenvironment, making 3D cultured tumor spheroids or organoids more effective models of in vivo tumors. They are particularly useful for small molecule drug screening and research into tumor-related molecular mechanisms, and can more accurately predict in vivo drug responses, efficacy, and toxicity. Additionally, 2D tumor cell models face limitations in expansion, often losing the genetic heterogeneity of the original tumor and exhibiting clonal selection, reducing clinical relevance. Compared to 2D cell models, 3D cell spheroids or organoids often provide more reliable research results and streamline drug evaluation processes. Since the first establishment of small intestine organoids in 2009, research on 3D cells and organoids has expanded to many tissue systems and become one of the hottest fields in life sciences.

Based on the requirement of 100μl of SBS3D™ Annexin V-PE Detection Working Solution per well in a 96-well plate, the small package of this kit can test 20 samples, the medium package can test 50 samples, and the large package can test 100 samples.

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 plates, Matrix-Gel or Matrigel-coated plates, agarose-coated plates, hanging drop culture plates, etc.
• The kit is easy to use, with the entire detection process taking approximately 15-30 minutes. After inducing apoptosis or necrosis in 3D cell spheroids, prepare the SBS3D™ Annexin V-PE Detection Working Solution by diluting SBS3D™ Annexin V-PE (20X) with SBS3D™ Annexin V-PE Binding Buffer in appropriate proportions, incubate in the dark for 15 minutes, and proceed with fluorescence microscopy imaging and analysis.

Components

• SBS3D™ Annexin V-PE (20X)
• SBS3D™ Annexin V-PE Binding Buffer

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Storage

Store at 4ºC for six months. Store at -20ºC for one year. SBS3D™ Annexin V-PE (20X) should be stored protected from light.

Precautions

• Although tests have shown that repeated freeze-thaw cycles (up to five times) of SBS3D™ Annexin V-PE (20X) do not significantly affect its detection performance, it is recommended to store it at 4ºC for 3-6 months and avoid repeated freeze-thaw cycles for optimal results.
• Cell spheroids may deform or disperse under external forces. Perform PBS washing and liquid changes gently to avoid damaging or dispersing 3D cell spheroids.
• Different types of cell spheroids may have varying tolerances to apoptosis or necrosis inducers. After inducing apoptosis or necrosis in 3D cell spheroids, their morphology may change. Observe the cell spheroid morphology under a microscope before staining and consider selecting more intact cell spheroids for staining analysis.
• If bacterial or fungal contamination is present, it may severely affect detection results.
• Staining should be performed promptly as prolonged time may lead to an increase in apoptotic or necrotic cells.
• Fluorescent substances are prone to quenching. Minimize observation time during fluorescence observation and store and handle samples protected from light.
• If trypsin was used during cell collection, ensure to remove residual trypsin, as it may digest and degrade SBS3D™ Annexin V-PE, leading to staining failure.
• Due to variations in cell samples and differences in levels of apoptosis and necrosis, optimize the amount of Annexin V-PE based on preliminary experiment results, adjusting as necessary.
• This product is intended for scientific research use by professionals only and is not for clinical diagnosis or treatment, food, or drugs. Do not store in residential areas.