Cell Apoptosis, which is a process of programmed cell death that occurs in multicellular organisms.Biochemical events lead to characteristic cell changes (morphology) and death. In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is a highly regulated and controlled process that confers advantages during an organism's life cycle. It will be easy to distinguish via morphology, biochemistry and molecular biology differences.
Morphological detection of cell apoptosis
1.Optical microscopy and inverted microscope
A.Unstained cells: the volume of apoptotic cells becomes smaller, deformed, the cell membrane is complete but foaming occurs, and apoptotic bodies are seen in the late stage of apoptosis. Adherent cells appear shrinkage, turn round, fall off.
B. Dyeing cells: commonly used Giemsa staining, Wright's staining. Apoptotic cells, chromatin condensation, marginalization, nuclear membrane lysis, chromatin is divided into massive and apoptotic bodies and other typical apoptotic morphology.
2.Fluorescence microscopy and confocal laser scanning microscopy
Generally, the morphological changes of nuclear chromatin are used as indicators to evaluate the progress of apoptosis. Commonly used DNA-specific dyes are: HO 33342 (Hoechst 33342), HO 33258 (Hoechst 33258), DAPI. The combination of the three dyes with DNA is non-embedded and is predominantly bound to the A-T base region of DNA. The UV light emits bright blue fluorescence while excitation. As a result, The morphological changes of nuclear chromatin during the process of apoptosis were divided into three phases: the nucleus of the stage Ⅰ was rippled or creased, and some chromatin appeared in the concentrated state. The chromatin height of the nucleus of stage Ⅱa Coagulation, marginalization; Ⅱ b phase of the nuclear cell lysis, resulting in apoptotic bodies.
Annexin V method
Phosphatidylserine (PS) is normally located inside the cell membrane, but at the early stage of apoptosis, PS can be flipped from the inside of the cell membrane to the surface of the cell membrane and exposed to the extracellular environment (Figure 3). Annexin-V is a Ca2 + -dependent phospholipid-binding protein with a molecular weight of 35-36 KD, which binds specifically to PS affinity. Annexin-V was labeled with fluorescein (FITC, PE) or biotin. Annexin-V was used as the fluorescent probe. The apoptosis was detected by flow cytometry or fluorescence microscopy.
Detection of Potential Energy of Mitochondrial Membrane
Mitochondria play a pivotal role in the process of apoptosis, a variety of apoptosis stimulating factors can induce apoptosis of different cells, and mitochondrial transmembrane potential (Δψm) decline, is regarded as the earliest event in the process of apoptosis cascade, if it occurred in the cell apoptosis characteristics (chromatin condensation, DNA cleavage) before the occurrence of mitochondrial transmembrane potential collapse, then the apoptosis is irreversible.
DNA fragmentation detection
The main biochemical characteristic of apoptosis is that the chromatin is concentrated and the chromatin DNA breaks at the junction between the nucleosomes, forming a large DNA fragment of 50 to 300 kbp or an integer multiple of 180 to 200 bp The nucleotide fragments were expressed as ladder electrophoresis (DNA ladder) on gel electrophoresis.
After the cells were treated, the purified DNA was isolated and purified by conventional methods, and subjected to agarose gel electrophoresis and ethidium bromide staining. Typical DNA ladder was observed in the apoptotic cell population. If the amount of cells is small, DNA can be labeled with 32P-ATP and deoxyribonucleotide terminal transferase (TdT) after separation and purification of DNA, followed by electrophoresis and autoradiography to observe the formation of DNA ladder in apoptotic cells.
TdT-mediated dUTP Nick-End Labeling
In the process of apoptosis, the chromosomal DNA double-strand breaks or single-strand breaks produce a large amount of viscous 3'-OH terminus, which can react with deoxyribonucleotides and fluorescence under the action of deoxyribonucleotide terminal transferase (TdT) Derivatives of peroxidase, alkaline phosphatase or biotin are labeled to the 3 & apos; -end of the DNA so that apoptosis can be detected.
TFAR19 Protein Expression and Cell localization analysis
Taking the fluorescein (FITC) -labeled TFAR19 monoclonal antibody as a probe to study on the expression level and localization of TFAR19 protein in the process of apoptosis. The expression level of TFAR19 in the early stage of apoptosis getting higher, accompanied with changes in the morphology of the nucleus, and still can be seen in the apoptotic bodies for a long time!
All mentioned above are the methods can be used to determine cell apoptosis. Besides, there are still some other ways can be used for cell apoptosis determination.