Common Methods for Cell Apoptosis Detection: Principles, Applications and Absin Premium Solutions

1. Overview of Cell Apoptosis

Cell apoptosis is an active and ordered form of cell death that involves the activation, expression, and regulation of a series of genes. It is not a phenomenon of autologous damage under pathological conditions, but an active form of death striving to better adapt to the living environment.

2. Apoptosis-Related Signaling Pathways

Apoptosis signaling pathways mainly include Caspase-dependent cell apoptosis and Caspase-independent cell apoptosis, which are further divided into specific sub-pathways:

•         Caspase-dependent cell apoptosis:
       

￮       Extrinsic apoptosis: Death receptor pathway.

￮       Intrinsic apoptosis: a. Endoplasmic reticulum stress pathway; b. Mitochondrial pathway.

•         Caspase-independent cell apoptosis:
       

￮       Cell death induced by Bcl family protein Bax.

￮       Other proteases, such as calpains, proteasomes, and serine proteases-mediated cell apoptosis.

3. Morphological Changes of Apoptosis

The morphological observation of apoptosis changes is multi-staged, and apoptosis often involves multiple cells, even a small number of cells occur asynchronously. The first change is cell shrinkage, loss of connections, and detachment from surrounding cells, followed by increased cytoplasmic density. A series of subsequent changes include: disappearance of mitochondrial membrane potential; altered permeability; release of cytochrome C into the cytoplasm; nuclear chromatin condensation, nuclear membrane and nucleolus fragmentation; DNA degradation into approximately 180bp-200bp fragments, formation of small vesicles on the cell membrane; phosphatidylserine on the inner side of the membrane flips to the membrane surface; the cell membrane structure remains intact, and finally, the remains of apoptotic cells can be divided and wrapped into several apoptotic bodies without overflow of contents.

4. Common Methods for Cell Apoptosis Detection

Common methods for cell apoptosis detection include morphological observation, DNA gel electrophoresis, enzyme-linked immunosorbent assay (ELISA) for nucleosome determination, flow cytometry quantitative analysis, Western blot detection, and more. Detailed introductions to each method are as follows:

4.1 Morphological Observation

Electron microscopy observation is a classic approach for morphological observation of apoptosis. Under electron microscopy, apoptotic cells exhibit disappearance of surface microvilli, condensation and margination of nuclear chromatin (often crescent-shaped), nuclear membrane folding, compact cytoplasm, aggregation of organelles, cell membrane blebbing or "budding", formation of apoptotic bodies, and phagocytosis of apoptotic bodies by adjacent phagocytes. This method provides intuitive and accurate morphological evidence of apoptosis, but it requires professional equipment and technical operations, and is not suitable for high-throughput detection.

4.2 DNA Gel Electrophoresis

A major biochemical characteristic of apoptosis is chromatin condensation, and chromatin DNA breaks at the junctions between nucleosome units, forming oligonucleotide fragments that are integer multiples of 180-200bp. These fragments present a ladder-like electrophoretic pattern (DNA ladder) in gel electrophoresis. The detection process involves DNA extraction, electrophoresis, and observation of the DNA ladder under UV light. This is a simple method to determine the occurrence of apoptosis, and is particularly suitable for the detection of non-proliferating cells cultured in vitro. However, it has limitations in detecting early apoptosis and requires a sufficient number of apoptotic cells to obtain obvious results.

Lane 1: DNA ladder; Lanes 2-3: Necrosis; Lanes 4-6: Apoptosis; Lane 7: Normal cells.

4.3 Enzyme-Linked Immunosorbent Assay (ELISA) for Nucleosome Determination

DNA fragmentation during apoptosis leads to the appearance of nucleosomes in the cytoplasm. Nucleosomes are composed of histones and their associated DNA fragments, which can be detected by ELISA. This method has the advantages of high sensitivity, wide application range, and simple operation, but it cannot achieve precise quantification.

4.3.1 Detection Steps

1.       Lyse apoptotic cells and centrifuge at high speed; the supernatant contains nucleosomes.

2.       Adsorb histones on a microtiter plate.

3.       Add the supernatant to allow anti-histone antibodies to bind to histones on nucleosomes.

4.       Add horseradish peroxidase-labeled anti-DNA antibodies to bind to DNA on nucleosomes.

5.       Add enzyme substrate and measure the light absorption value.

4.4 Annexin V/PI Assay

In normal cells, phosphatidylserine (PS) is located on the inner side of the cell membrane. However, in early apoptotic cells, PS flips from the inner side of the cell membrane to the surface, exposing to the extracellular environment. Annexin-V is a Ca²⁺-dependent phospholipid-binding protein with a molecular weight of 35-36KD, which can bind to PS with high affinity. Using Annexin-V labeled with fluorophores (such as FITC, Alexa Fluor488) as a probe, the occurrence of apoptosis can be detected by flow cytometry or fluorescence microscopy.

Propidium iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane. However, in mid-to-late apoptotic cells and necrotic cells, PI can penetrate the cell membrane and stain nuclear DNA. Therefore, the combined use of Annexin-V and PI can distinguish early apoptotic cells and late apoptotic cells in a cell population:

•         Quadrant B3: Annexin V⁻/PI⁻: Viable cells;

•         Quadrant B4: Annexin V⁺/PI⁻: Early apoptotic cells;

•         Quadrant B2: Annexin V⁺/PI⁺: Late apoptotic cells;

•         Quadrant B1: Annexin V⁻/PI⁺: Necrotic cells.

This method is widely used due to its simplicity, rapidity, and ability to quantify, and is suitable for various types of cells. Absin provides high-quality Annexin V-FITC/PI Apoptosis Detection Kit (Catalog No.: abs50001) to support related research.

4.5 TUNEL Assay

During apoptosis, double-strand breaks or single-strand breaks of chromosomal DNA produce a large number of sticky 3'-OH ends. Deoxynucleotide derivatives conjugated with fluorophores, peroxidase, alkaline phosphatase, or biotin can be labeled to the 3' ends of DNA under the action of terminal deoxynucleotidyl transferase (TdT). This type of method is called terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).

Common labeling systems: Biotin-labeled dUTP, digoxigenin-labeled dUTP, fluorophore-labeled dUTP, bromine-labeled dUTP;

Common chromogenic systems: Anti-biotin/digoxin/biotin-HRP and DAB; FITC-dUTP/BrdU and flow cytometry.

The TUNEL assay is suitable for detecting apoptotic cells in tissue sections and cell samples, and can accurately locate apoptotic cells, but it may have false positives in cells with DNA damage.

4.6 Western Blot Detection

This method analyzes the activation of Procaspase-3, as well as the cleavage of activated Caspase-3 and its substrate poly (ADP-ribose) polymerase (PARP). The Caspase family plays a crucial role in mediating apoptosis, among which Caspase-3 is a key executor that functions in many pathways of apoptotic signal transduction.

Caspase-3 normally exists in the cytoplasm as a zymogen (32KD). It is activated in the early stage of apoptosis, and the activated Caspase-3 consists of two large subunits (17KD) and two small subunits (12KD), which cleave corresponding cytoplasmic and nuclear substrates, ultimately leading to cell apoptosis. However, in the late stage of apoptosis and dead cells, the activity of Caspase-3 decreases significantly.

Absin provides high-quality antibodies for Western blot detection of apoptosis-related proteins, such as Caspase-3 Antibody (Catalog No.: abs131825) and Cleaved Caspase-3 (Asp175) Antibody. These antibodies have been validated in experiments: Western blot analysis of HeLa, NIH/3T3, and C6 cell extracts untreated, treated with staurosporine (3 hours, 1 µM, in vivo) or treated with cytochrome c (1 hour, 0.25 mg/ml, in vitro) using abs131825 - Caspase-3 Antibody (upper) or Cleaved Caspase-3 (Asp175) Antibody (lower) yields clear and reliable results.

4.7 Fluorescence Spectrophotometer Analysis

Activated Caspase-3 can specifically cleave the D1E2V3D4-X substrate and hydrolyze the D4-X peptide bond. Based on this characteristic, a short peptide Ac-DEVD-AMC (Caspase-3 tetrapeptide fluorescent substrate) conjugated with a fluorescent substance is designed. In the covalently conjugated state, AMC cannot be excited to emit fluorescence; after the short peptide is hydrolyzed, AMC is released, and free AMC can be excited to emit fluorescence. The activity of Caspase-3 can be determined according to the intensity of the released AMC fluorescence, thereby reflecting the activation degree of Caspase-3.

Experimental Example: Hela cells were cultured in 96-well plates and incubated overnight. Cells were treated with different concentrations of Staurosporine #9953 for 5 hours, then lysed. The cell lysate was mixed with the substrate solution and incubated at 37ºC in the dark for 2 hours to obtain relative fluorescence units (RFU).

4.8 Analysis of Other Apoptosis-Related Factors

4.8.1 Dissipation of Mitochondrial Transmembrane Potential (△Ψm)

Mitochondrial transmembrane potential is closely related to apoptosis. Specific fluorescent dyes that are sensitive to mitochondrial membrane potential include TMRM (emitting red fluorescence), cell membrane fluorescent probe DiOC6 (3), iodide (emitting green fluorescence), Rhodamine 123 (emitting yellow-green fluorescence), and JC-1 (monomer form emits green fluorescence; polymer form emits red fluorescence). The increase or decrease of their fluorescence indicates the increase or decrease of the electronegativity of the inner mitochondrial membrane. Absin provides Mitochondrial Membrane Potential Detection Kit (JC-1) (Catalog No.: abs50016-100T) to facilitate the detection of mitochondrial transmembrane potential changes.

4.8.2 Detection of Changes in Intracellular Redox State

The decrease of glutathione in the cytoplasm of apoptotic cells can be detected in vitro by the fluorescent dye Monochlorobimane (MCB) to reflect the changes in the intracellular redox state in the early stage of apoptosis.

4.8.3 Detection of ROS Produced by Mitochondria

Non-fluorescent hydroethidine (HE) can be oxidized by ROS to ethidium bromide (EthBr), which emits red fluorescence. NAO (alkylacridine orange, which can emit fluorescence) can bind to non-oxidized cardiolipin (which can be oxidized by ROS) and thus lose fluorescence. These dyes can be used to detect ROS produced by mitochondria during apoptosis.

5. Absin Recommended Products for Cell Apoptosis Detection

To meet the diverse needs of cell apoptosis research, Absin, a sub-brand of AN BIO PTE. LTD., provides a full range of high-quality apoptosis detection reagents covering various detection methods. The recommended products are as follows:

6. About AN BIO PTE. LTD.

AN BIO PTE. LTD. is committed to providing high-quality, reliable biological research reagents and comprehensive solutions for global scientific researchers. With its professional sub-brand Absin, we focus on the research and development and production of products related to cell biology, immunology, and molecular biology. Our products are widely used in academic research, drug development, and clinical diagnosis, winning the trust and recognition of numerous researchers.

We adhere to the core values of "quality first, customer-oriented", continuously optimize product performance and service quality, and strive to become a trusted partner in the field of life sciences, contributing to the progress of global biomedical research.

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