Common methods for detecting apoptosis in cells

Common methods for detecting apoptosis in cells

Apoptosis, also known as programmed cell death, is an orderly or programmed cellular death process regulated by specific genes. It is a proactive death process of cells and a normal physiological response of cells. Apoptotic cells are ultimately processed by phagocytes in the body. Excessive apoptosis directly leads to a decline in the function of tissues and organs; whereas, for cancer cells, interruption of the apoptosis process signifies the development and spread of cancer.

Morphological Changes of Cells

It can usually be divided into three stages. The first stage involves the disappearance of cell-to-cell contact, with intact cell membranes. Ribosomes detach from the rough endoplasmic reticulum, and the nucleus shows signs of condensation. This stage lasts for a very short period. The second stage involves nuclear fragmentation, where the fragmented nucleus combines with various organelles inside the cell and is further enveloped by the cell membrane to form apoptotic bodies. The third stage involves phagocytic cells engulfing apoptotic bodies and digesting them for reuse by the organism.


Mechanisms of Cell Apoptosis

(1) Death Receptor Pathways and Regulation: Fas/Fas ligand (FasL) death pathway; TNF receptor (TNFR) death pathway.

(2) Mitochondrial Pathway and Regulation.

(3) Endoplasmic Reticulum Pathway: CHOP pathway; JNK pathway; Caspases 12 pathway.

(4) Telomerase Pathway of Cell Apoptosis.

(5) NO-involved Cell Apoptosis Response.


Common methods for detecting cell apoptosis include the following

1. Morphological Detection

Cells undergoing apoptosis exhibit specific morphological characteristics. The most commonly used morphological detection methods are fluorescence confocal laser scanning microscopy and transmission electron microscopy. The former allows specific dyes (such as Hoechst 33342, Hoechst 33258, DAPI) to stain cells. Non-intercalating binding to the A-T base region of DNA emits bright blue fluorescence when excited by ultraviolet light.

Morphological Changes of Nuclear Chromatin in DAPI Staining of Apoptotic Hela Cells

During apoptosis, the nuclei of Hela cells undergo morphological changes. Concurrently, apoptotic cells shrink in size, and their cytoplasm becomes more condensed. In the early stages of apoptosis (pro-apoptosis nuclei), chromatin within the cell nucleus is highly condensed and exhibits numerous vacuole-like structures known as cavitations. In the intermediate stage (Ⅱa phase), the chromatin within the cell nucleus becomes highly condensed and marginalized. In the late stages of apoptosis, the cell nucleus fragments into smaller pieces, forming apoptotic bodies. Under electron microscopy, apoptotic cells show decreased volume, disappearance of surface microvilli, and condensed cytoplasm.

Morphological Changes of Nuclear Chromatin in Apoptotic Jurkat Cells Under Electron Microscopy 

2.The most commonly used method for detecting early apoptosis is the Annexin V / PI double staining assay.

In normal cell membranes, the distribution of phospholipids is asymmetrical, with phosphatidylserine (PS) located on the inner surface of the cell membrane. During cell apoptosis, changes occur in the cell membrane, causing PS to flip from the inner to the outer surface of the cell membrane. Annexin V is a calcium-dependent phospholipid-binding protein with a high affinity for PS. Annexin V can specifically recognize PS on the surface of apoptotic cells, as PS in live cells is located on the inner surface of the cell membrane and cannot bind specifically to Annexin V. Therefore, FITC-conjugated Annexin V can be used to distinguish apoptotic cells from live cells.

The PS of necrotic cells also flips from the inner to the outer surface of the cell membrane, and Annexin V can also recognize PS on the surface of necrotic cells. Therefore, Annexin V cannot distinguish between necrotic cells and apoptotic cells. However, propidium iodide (PI) dye can bind to DNA inside cells, distinguishing between necrotic cells and live cells. In early apoptotic cells and live cells, the cell membrane remains intact, preventing PI dye from freely passing through the cell membrane to bind to DNA inside the cell. Therefore, PI dye cannot label apoptotic cells and live cells. PI dye can, however, pass through the cell membrane of necrotic cells and bind to DNA inside the cell. When PI dye inside necrotic cells is excited by a 488nm laser, it emits red fluorescence, which is detected by the corresponding channel. Therefore, by using Annexin V and PI simultaneously, it is possible to distinguish between live cells, early apoptotic cells, and necrotic cells.

3.Cell Apoptosis - Detection of Mitochondrial Membrane Potential

JC-1 is a lipophilic cationic fluorescent dye that can bind to the mitochondrial matrix. The enhancement or reduction of its fluorescence indicates an increase or decrease in the electronegativity of the mitochondrial membrane. Under normal physiological conditions, the mitochondria have a high negative charge, and JC-1 enters the mitochondria in its aggregated form, emitting red fluorescence strongly. During cell apoptosis, mitochondrial depolarization occurs, leading to a decrease in negativity. Consequently, JC-1 exists as monomers in the cytoplasm, resulting in enhanced green fluorescence.


4.Cell Apoptosis - Detection of Activated Caspase-3

The Caspase family plays a crucial role in mediating the process of cell apoptosis, with Caspase-3 being a key executioner molecule that functions in many pathways of apoptosis signal transduction. Caspase-3 exists in the cytoplasm as a proenzyme (32 KD). In the early stages of apoptosis, it becomes activated. Activated Caspase-3, composed of two large subunits (17 KD) and two small subunits (12 KD), cleaves corresponding cytoplasmic and nuclear substrates, ultimately leading to cell apoptosis. However, in the late stages of apoptosis and in dead cells, the activity of Caspase-3 significantly decreases.

Activated Caspase-3, formed by the cleavage of its precursor into a 17KD subunit and a 12KD subunit, forms a heterodimer, which can be specifically recognized by Anti-Active Caspase-3 antibody. Detection of activated Caspase-3 can be achieved by labeling Anti-Active Caspase-3 antibody with fluorochrome.

5.Cell Apoptosis - Detection of DNA Fragmentation

During cell apoptosis, chromatin condenses, and the DNA within the chromatin breaks at the connections between nucleosome units, forming oligonucleotide fragments of 180~200bp and its multiples. Once DNA fragmentation occurs, numerous gaps appear on the DNA double-stranded structure. FITC-labeled dUTP or BrdU can be incorporated into these gaps under the action of enzymes, and then the intensity of the incorporated signal can be detected to determine the extent of fragmentation.

6.TUNEL Detection Method

Additionally, another method for detecting DNA fragmentation is the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) detection method. During cell apoptosis, the breakage of DNA double-strands or single-strands produces a large number of sticky 3’-OH ends. Using this as a cue, with the assistance of Terminal deoxynucleotidyl Transferase (TdT), biotin/fluorescein-labeled dUTP is successfully bound to the 3’-OH ends. The results can be quantitatively analyzed through enzyme-linked colorimetric or fluorescence detection.

In summary, cell apoptosis is facilitated by various molecules in apoptotic pathways, and apoptosis occurrence cannot be easily determined solely based on the detection of a single indicator. After all, in events of cell apoptosis, each indicator is only maintained for a certain period. Therefore, it is advisable for researchers to cast a wide net and employ multiple indicators, preferably at different time points, to confirm the occurrence of apoptosis. Finally, to avoid false positives and false negatives, positive and negative controls are essential to ensure the accuracy of the detection results.


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