CELL BIOLOGY ACADEMY | ULTRA-DETAILED APOPTOSIS ASSAY SELECTION GUIDE!

Apoptosis, a well-studied form of programmed cell death, has emerged as a focal point in biological research in recent years. If your work involves cancer mechanisms and therapies, drug screening and pharmacodynamics, developmental biology, immunology, or neuroscience, apoptosis detection is undoubtedly a critical component of your experimental design.

In this edition of Cell Biology Academy, we will provide an in-depth overview of different types of apoptosis detection kits and share practical insights on how to select the most suitable kit for your samples. Our goal is to help you easily understand and make informed choices for your apoptosis detection needs.

Decoding Apoptosis: Insights into Detection Methods

Apoptotic cells exhibit distinct morphological and physiological differences from necrotic and healthy cells. Notable alterations occur in the cell membrane, cytoplasm, nucleus, nucleic acids, and apoptosis-related proteins, providing clear distinguishing features. These variations enable the development of multiple methods for detecting apoptosis. Common techniques for apoptosis detection include morphological assessment, phosphatidylserine externalization assays, detection of apoptosis-related proteins, DNA fragmentation analysis, and cell cycle profiling. Each method has its unique characteristics, allowing researchers to choose the most suitable approach based on experimental requirements and conditions.

Based on the mechanisms and characteristics of apoptosis, commonly available detection kits on the market include Annexin V assays, TUNEL assays, Caspase assays, and Mitochondrial Membrane Potential (JC-1) assay kits.

Annexin V Apoptosis Assay Kit

Detection principle: Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). In normal cells, PS is usually located on the inside of the cell membrane. However, when apoptosis occurs, the permeability of the cell membrane changes, causing the PS on the inner side to turn outward and become exposed on the outer side of the membrane. Annexin V can recognize apoptotic cells by binding to PS in cells with cell eversion in the early stages of apoptosis. To facilitate detection, Annexin V is labeled with a fluorescent dye that allows fluorescent apoptotic cells to be detected by equipment such as flow cytometry or fluorescence microscopy. Nuclear dyes (PI, DAPI, 7-AAD) bind specifically to double-stranded DNA and produce intense fluorescence. Under normal circumstances, macromolecular nuclear dyes are impermeable to cell membranes, but due to the loss of integrity of the necrotic cell membrane in the late stage of apoptosis or necrosis, the nuclear dye can enter the cell to stain DNA, and can be used with Annexin V to distinguish cells in different apoptotic stages.

TUNEL Apoptosis Assay Kit

Detection principle: In the late stage of apoptosis, chromosomal DNA double-strand breaks produce a large number of sticky 3'-OH ends. Under the action of terminal deoxynucleotidyl transferase (TdT), derivatives formed by deoxyribonucleotide and markers such as fluorescein can be labeled to the 3'-end of the broken DNA for the detection of apoptotic cells.

Caspase Activity Assay Kit

Detection principle: Caspase is a family of proteases that are usually present in the cytoplasm in the form of inactive zymogens. Activated caspase destroys some important intracellular proteins, leading to the occurrence of apoptosis. A caspase sequence-specific polypeptidase is conjugated to the yellow group pNA as a substrate, and when the caspase is activated, it is able to cleave this substrate and release a free yellow group. By measuring the absorbance of these yellow groups by microplate reader or spectrophotometer, the activity of Caspase can be quantitatively assessed and thus the apoptosis can be indirectly monitored.

Mitochondrial membrane potential (JC-1) assay kit

Detection principle: In normal cells, the mitochondrial membrane potential is usually high, and JC-1 mainly aggregates in the mitochondrial matrix, exists in the form of aggregates, and can produce red fluorescence. However, in the early stages of apoptosis, the mitochondrial membrane potential decreases significantly, resulting in the inability of JC-1 to accumulate in the mitochondrial matrix and to exist mainly in monomeric form, which can produce green fluorescence. The decrease of cell membrane potential can be detected by the transition of JC-1 from red fluorescence to green fluorescence, and the transition of JC-1 fluorescence color can be used as a detection indicator for early apoptosis.

How to choose an apoptosis assay

There are many types of apoptosis assays currently on the market, and it is important to choose a kit that matches your test sample and experimental conditions. Here are two suggestions:

1. Selection based on sample type

Select the appropriate apoptosis assay based on your specific experimental design and sample type. It is recommended that you refer to the table below to select the appropriate apoptosis assay for your sample type. For example, suspension cells or digested adherent cells, all four apoptosis kits can be considered, which can often be adapted to different types of cell samples. However, for tissue samples, it is important to note that different kit sample preparation methods may differ. The TUNEL Apoptosis Assay is prepared with paraffin or frozen sections, while with the Caspase Activity Assay, the tissue is mechanically minced, homogenized, and lysed.

2. Selection based on experimental instrument and configuration

The use of different apoptosis detection kits requires the appropriate instrumentation. It is recommended that you refer to the table below to select the appropriate apoptosis detection kit according to the existing experimental instruments in the laboratory. It should be reminded that in addition to confirming whether the instrument is available, it is also necessary to understand the specific configuration of the experimental instrument, such as the fluorescence detection channel of the flow cytometer and the filter set of the fluorescence microscope.

Commercially available Annexin V apoptosis assays and TUNEL apoptosis assays are available in a variety of fluorochrome combinations. When selecting a fluorochrome, it is necessary to select the fluorochrome that can be detected according to the instrument configuration. In addition, if your cells are self-fluorescent (e.g., cells transfected with fluorescent proteins or cells treated with fluorescent drugs), preferably fluorescent dyes that are different from the cells autofluorescence if the instrument configuration allows. If the fluorochrome spectral overlap cannot be avoided due to the configuration conditions of the instrument, it is necessary to pay attention to the reasonable setting of a single dye tube to adjust the compensation to ensure the accuracy of the detection results.

Information about apoptosis assay kits

The above is a guide to the knowledge and selection of apoptosis detection kits, for more dry goods related to cell culture and detection, please continue to pay attention to the column of Cell Biology Academy.