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

Cat. No.: 3DAVMS-50 (for 50T)

Description

SBS3D™ Annexin V-mCherry/SYTOX Green Apoptosis Detection Kit uses the apoptosis fluorescent probe Annexin V-mCherry combined with the dead cell green fluorescent probe SYTOX Green to simultaneously detect apoptotic and necrotic cells in 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, and green fluorescence in the nuclei of necrotic cells under a fluorescence microscope. This kit is suitable for use with fluorescence microscopes, fluorescence microplate readers, and other fluorescence detection systems.

This kit detects the red fluorescence of Annexin V-mCherry and the green fluorescence of SYTOX Green. The maximum excitation wavelength of Annexin V-mCherry is 587nm, and the maximum emission wavelength is 610nm. When intercalated into DNA, the maximum excitation wavelength of SYTOX Green is 504nm, and the maximum emission wavelength is 523nm.

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.

SYTOX Green is a non-cell membrane permeable cyanine dye. It cannot pass through the intact cytoplasmic membrane of viable cells but can pass through disordered regions of the membrane in dead cells and enter the nucleus. When intercalated into DNA, SYTOX Green forms a complex that increases fluorescence intensity over 500-fold, producing bright green fluorescence. Therefore, SYTOX Green stains only dead cells, allowing differentiation between normal and necrotic cells.

Compared to Annexin V labeled with phycoerythrin (PE), Annexin V-mCherry has a narrower excitation spectrum and does not produce red fluorescence around 495nm like PE, making it ideal for simultaneous detection with other green fluorescent proteins.

Normal cells will not be stained by Annexin V-mCherry or SYTOX Green. Apoptotic and necrotic cells will be stained by Annexin V-mCherry, and the nuclei of necrotic cells will be stained bright green by SYTOX Green. It has been reported that the nuclei of mid-to-late apoptotic cells can be stained weak green by SYTOX Green.

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-mCherry/SYTOX Green Detection Working Solution per well in a 96-well plate, the small package of this kit can test 20 samples, and the medium package can test 50 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-mCherry/SYTOX Green Detection Working Solution by diluting SBS3D™ Annexin V-mCherry (40X) and SBS3D™ SYTOX Green Staining Solution (100X) with SBS3D™ Annexin V-mCherry Binding Buffer in appropriate proportions, incubate in the dark for 15 minutes, and proceed with fluorescence microscopy imaging and analysis.

Components

• SBS3D™ Annexin V-mCherry (40X)
• SBS3D™ Annexin V-mCherry Binding Buffer
• SBS3D™ SYTOX Green Staining Solution (100X)

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Storage

Store at -20ºC for one year. SBS3D™ Annexin V-mCherry (40X) and SBS3D™ SYTOX Green Staining Solution (100X) should be stored protected from light.

Precautions

• Repeated freeze-thaw cycles may reduce staining efficacy. To ensure optimal use, avoid repeated freeze-thaw cycles and consider aliquoting after the first thaw for storage.
• 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-mCherry, leading to staining failure.
• 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.
• For your safety and health, wear a lab coat and disposable gloves while handling.