Introduction to apoptosis detection methods

Apoptosis refers to the cell's growth to a certain stage and certain physiological processes, which are subject to strict regulation by multiple factors and mechanisms and enter death. This is a dynamic process involving a complex series of biochemical reactions, a cascade of reactions that require enzymes, as well as the expression and regulation of different genes, and signaling. Several physiological processes can be used to detect the occurrence of apoptosis, so multiparametric analysis is important for accurate detection of this process. At the same time, understanding cell death and survival mechanisms is also a key component of toxicology analysis and drug development applications.

(A) Caspase activity detection

Caspase (cysteinyl-aspartic acid proteases) is a family of proteases whose activation is a typical feature of apoptosis. Once a Caspase enzyme is activated, a series of other Caspas enzymes are activated, cleavage of the corresponding cytoplasmic nucleus substrate, which ultimately leads to apoptosis. Caspase 3 activation occurs early in apoptosis and plays an important role in the process of apoptosis. Caspase-Glo®3/7 Assay provides a homogeneous luminescence assay for detecting Caspase-3/7 activity.

(2) Membrane change detection

Another biochemical feature of apoptotic cells is the transfer of phosphatidylserine from the cytoplasm to the extracellular. Annexin V in annexin is a phospholipid-binding protein with strong affinity for phosphatidylserine. Early detection of apoptotic cells can be performed by using Annexin V FLUOS Staining Kit or Annexin V-FITC/PI Apoptosis Detection Kit. Cytometry or fluorescence microscopy. At the same time, PI is used to distinguish early apoptotic cells and necrotic cells according to the permeability of the cell membrane.

Changes in mitochondrial membrane potential and membrane permeability are also important features of apoptotic cells. The apoptotic cell mitochondria can be isolated using the Mitochondria Isolation Kit, and the membrane potential can be detected by the Mitochondrial membrane potential assay kit (JC-1) to detect early apoptosis.

(III) DNA fragmentation analysis

DNA degradation is also a typical marker of apoptosis, and its degradation products can be divided into two types, namely high molecular weight single-stranded DNA gaps and low molecular weight DNA fragments (single-stranded and oligonucleosomes).

Single-stranded DNA gaps have a specific detection method that uses enzyme catalysis to label specific nucleic acids to the free 3'-OH end. This method is called TUNEL. The DeadEndTM Colorimetric TUNEL System from Promega is a TUNEL assay that enables easy, rapid and accurate in situ detection of apoptotic cells at the single cell level.

A histone-associated DNA fragment (single-stranded and oligosome) that appears in the cytoplasm of apoptotic cells can be detected by high-throughput cell death using the Cell Death Detection ELISA PLUS or purified using the Apoptotic DNA-Ladder Detection Kit. And agarose electrophoresis analysis of these DNA fragments exhibiting a typical ladder state.

(four) cell morphology test

Changes in cell morphology such as membrane wrinkles or blistering, dense nuclear chromatin, and cytoplasmic condensation can be used as evidence of apoptosis. The progression of apoptosis is generally judged by the morphological changes of nuclear chromatin as an indicator. Commonly used DNA-specific dyes are: Hoechst 33342, Hoechst 33258, DAPI. The combination of the three dyes and DNA is non-embedded and mainly binds to the AT base region of DNA. Bright blue fluorescence is emitted when excited by ultraviolet light.

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