Apoptosis detection: a purpose-dependent approach selection

ABSTRACT

Apoptosis is closely associated with many diseases. Detection of apoptosis can be achieved by morphology, biochemistry, molecular biology, immunology, and other techniques. However, as technologies are increasingly used for the detection of apoptosis, many researchers are confused about how to choose a suitable method to detect apoptosis. Selection of a suitable detection method for apoptosis will help clinical diagnosis and prevention of diseases. This article reviews the selection of optimal apoptosis-detection methods based on research purposes and technique principles.

1. Introduction

Apoptosis is important in the maintenance of body hemostasis in tissue differentiation, organ development and aging, injury, and elimination of mutant cells [1]. Later, Wyllie et al. showed that apoptotic cells have morphological features, such as cell shrinkage, chromatin agglutination, apoptotic body formation, and DNA fragmentation, which is related to endogenous endonuclease activation [2]. Vaux et al. then demonstrated that the B-cell lymphoma 2 gene (Bcl-2) plays a role in inhibiting apoptosis [3], and Miura et al. initiated research into the activation of apoptotic proteases [1]. There are many methods and technologies available for the detection of apoptosis. Thus, it is important to know how to choose an appropriate method for the detection of apoptosis. This review discusses how to choose the optimal apoptosis-detection method based on research purposes, principles, advantages and disadvantages of individual methods, and scope of the application. This paper provides a source for researchers to screen and identify the appropriate detection method for apoptosis for their research needs.

1.1. Morphological characteristics of apoptosis

Apoptosis can be divided into 3 phases: Phases I, IIa, and IIb, according to the morphological characteristics of the nucleus [4]. In Phase I, apoptotic cells shrink and acquire a dense cytoplasm, with decreased water content, increased eosinophilia, and the disappearance of microvilli on the cell surface. In addition, these apoptotic cells are separated from the surrounding normal cell population. In Phase IIa, chromatin condensation occurs and chromatin becomes dense masses (pyknosis) or is assembled on the inner nuclear membrane (chromatin margination), and subsequently the nuclei are broken into fragments (fragmentation). In Phase IIb, the cytoskeleton degrades, causing invaginations in the cell membrane, or sprouting and displacement, which cause the formation of membrane-coated vesicles containing cytoplasmic membrane, nuclear debris, and organelle components that are transformed into small bodies (apoptotic bodies). These are presumably the important morphological markers of cells [5]. Due to the integrity of the plasma membrane, the contents of the apoptotic cell are not released. This prevents molecules on other cells from recognizing the cellular contents and triggering a peripheral resonance to cause mesenchymal cell proliferation and repairing.

1.2. Apoptosis-detection methods based on general morphological features

The changes in morphological features that occur during apoptosis can be identified via microscopy in order to determine whether the cells are apoptotic. Morphological features observed under a light microscope after hematoxylin and eosin (HE), Giemsa, or Wright’s staining include the shrinking, rounding, and shedding of nuclei, chromatin morphology, and apoptotic bodies of the cells. Morphological features observed under an electron microscope after uranyl acetate-lead citrate staining include decreased volume, concentrated cytoplasm, and formation of protrusions on the surface of apoptotic cells. Furthermore, transmission electron microscopy can reveal the ultra-morphological changes that occur in the cells at different stages of apoptosis. During Phase I of apoptosis, there are many vacuoles, namely cavitations, that appear in the cells. During Phase IIa of apoptosis, the chromatin is highly condensed and marginalized within the cells. During Phase IIb of apoptosis, the cell nuclei are lysed into fragments, producing apoptotic bodies [6]. Morphological features observed under fluorescence or confocal microscope after Hoechst 33,342, acridine orange (AO), or 4',6-diamidino-2-phenylindole (DAPI) staining, which indirectly reveals the nuclear and chromatin conditions of the cells according to the intensity and distribution of the fluorescence signals, determine the occurrence of apoptosis [7,8].

1.3. Advantages and disadvantages and the scope of application for different general morphological methods

The main advantages of observing morphological features and changes of apoptosis include the simplicity, convenience, intuition in the observation, and the acquisition of storable specimens for further study. Light microscopy reveals some surface marker molecules of cell debris, such as thrombospondin and adhesion glycoproteins, produced in apoptotic cells. These substances facilitate the recognition, phagocytosis, and clearance of the apoptotic cells by surrounding macrophages and other cells. However, the phagocytosis of apoptotic cells is very effective and rapid, and as such, apoptotic cells are quickly removed without leaving traces and causing any inflammatory responses. Thus, apoptosis in a small area is not easily recognized through morphology. Light microscopy is mainly suitable for the observation of cells in Phase IIb apoptosis [9]. Electron microscopy reveals typical apoptotic morphology and structure. However, not all cells have typical morphological features during apoptosis. Therefore, even if there is no typical apoptotic characteristics, we cannot rule out the occurrence of apoptosis, which needs to be determined in combination with other detection methods [6]. Electron microscopy is mainly suitable for the observation of Phases I, IIa, and IIb of apoptosis. Fluorescence or confocal microscopy combined with fluorescence labels directly reveal the changes in the nuclei of apoptotic cells. However, a small area of apoptosis is also not easily identified by observing the cellular morphology by fluorescence or confocal microscopy, which is mainly suitable for the observation of Phase IIb of apoptosis [10].

2.1. Biochemical characteristics of apoptosis

Under normal circumstances, intracellular aspartate-specific cysteine proteases (e.g. caspases and apoptotic proteases) and endonucleases exist in the cell nuclei as inactive zymogens that do not directly cause DNA fragmentation ^[11]. Apoptosis-inducing factors directly or indirectly increase the concentrations of Ca²⁺ and Mg²⁺ through signal transduction pathways that activate endogenous endonucleases. The activated endonucleases specifically act on the linker DNA regions of the nucleosome, breaking the nucleosome into oligonucleotides – either the length of a single nucleosome (180–200 bp) or multiple nucleosomes – which contain a hydroxyl group at the 3' end [11]. Apoptotic proteases and endonucleases, the main executors of apoptosis, cause protein degradation (cleavage) and DNA fragmentation that leads to apoptosis. DNA fragmentation is a characteristic biochemical indicator for the occurrence of apoptosis [12].

2.2. Molecular biological techniques based on the biochemical characteristics of apoptosis

DNA gel electrophoresis reveals the DNA fragments of 180–200 bp of chromosomal DNA and its integer multiples produced by the activated endogenous endonucleases in apoptotic cells. After the gel electrophoresis, the formation of characteristic ladder-like DNA bands is used to determine the occurrence of apoptosis [13]. An in situ DNA nick end labeling assay, i.e. terminal deoxynucleotidyl transferase (TdT) dUTP nick end-labeling (TUNEL) method, is based on the characteristic that the 3' end of DNA fragments of apoptotic cells contain hydroxyl groups [14], and these 3' end hydroxyl (OH) groups of the DNA fragments are labeled by TdT to determine the occurrence of apoptosis. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure the mRNA expression of apoptosis-related genes at different stages of apoptosis. In addition, analysis of mitochondrial membrane potential is used to detect the biochemical characteristics of apoptotic cells. The decrease in mitochondrial membrane potential is an early marker of apoptosis based on the mitochondrial pathway. By staining the cells and tissues with fluorescent lipophilic cationic dyes, an increase or decrease of fluorescence signals reflect the changes in electronegativity of the inner mitochondrial membrane [15].

2.3. Advantages and disadvantages of different molecular biology techniques and their application scope

DNA gel electrophoresis is simple to perform and a qualitatively accurate method but has poor specificity and sensitivity. It can only be used for the semi-quantitative detection of apoptosis, and it cannot localize the apoptotic cells. It is also not suitable for detecting slight damage to the DNA chain seen during the early stage of apoptosis. This method is only suitable for the observation of large-scale apoptotic cells observed during the middle and late stages of apoptosis [16]. In situ DNA nick end labeling using TdT to catalyze the binding of fluorescein isothiocyanate (FITC), biotin, or another labeled dUTP to the 3ʹ-OH group based on the principle of DNA fragmentation during apoptosis, which produces a large number of sticky 3ʹ-OH ends [17,18], is a relatively sensitive and specific method for counting and quantifying apoptotic cells, but this method also can yield false-positive results. Negative and positive controls should be set before the experiment is performed in order to ensure accurate findings. This method is suitable for the detection of late-stage apoptosis [13,19]. RT-qPCR or gene chip technology used for the measurement of apoptosis-related gene expression demonstrates the related gene and RNA expression levels at different stages of apoptosis [20,21], without involving the measurement of protein expression.

Certain dyes can be used for the analysis of mitochondrial membrane potential. When mitochondrial membrane potential is high, the dye aggregates within the mitochondrial matrix to form a polymer and produce red fluorescence; however, when mitochondrial membrane potential is low, the dye cannot accumulate in the mitochondrial matrix and appears as a monomer and produces green fluorescence [22]. The conversion of different fluorescent colors reflects the change in mitochondrial membrane potential of the cells. The relative ratio of red and green fluorescence is usually used to measure the ratio of mitochondrial depolarization, and green fluorescence is used to determine early apoptosis based on the mitochondrial pathway [23]. This detection method is suitable for revealing the changes in mitochondrial membrane potential during the early stage of apoptosis. The change in pH also affects the cell membrane potential. Thus, the pH of the dye solution should be consistent.

3.1. Signal transduction characteristics of apoptosis

The initial stage of apoptosis and the source of apoptosis signals are different. The mechanism of apoptosis is mainly divided into exogenous and endogenous pathways, including the exogenous death receptor signaling pathways, endogenous mitochondrial pathway, and endogenous endoplasmic reticulum pathways [24]. Taking endogenous mitochondrial pathway for example, apoptotic proteases, and endonucleases, which cause protein degradation (cleavage) and DNA fragmentation. The degree and ratio of apoptosis in cells can be determined by cell cycle detection. Overloaded intracellular Ca²⁺ triggers the activation of the mitochondrial-endoplasmic reticulum pathway, leading to apoptosis [25]. In addition, under normal circumstances, cytochrome C oxidase (Cyto-C) exists in the intermembrane space between the mitochondrial inner and outer membranes. Upon activation of the mitochondrial apoptotic pathway, Cyto-C is released from the mitochondria into the cytosol to initiate the caspase cascade. Thus, release of Cyto-C is a key step in the mitochondria-dependent apoptotic pathway [26]. Cyto-C subunit IV (COX4) is a membrane protein located on the mitochondrial inner membrane. When apoptosis occurs, COX4, a useful indicator of mitochondrial enrichment, is retained inside the mitochondria [27]. Measurement of Cyto-C and COX4 expression by western blotting in an enriched mitochondrial fraction isolated from the cells helps determine the occurrence of apoptosis. In addition, different factors can activate the endoplasmic reticulum and death receptor-mediated apoptosis pathways, and also lead to changes in the expression of related genes and proteins [28], so as to identify the signal transduction pathways that regulate apoptosis.

3.2. Detection of the apoptosis signal transduction pathway using immunological technique

The occurrence of apoptosis can be detected by the changes in the related target proteins of the apoptosis pathways. Immunolabeling technology refers to the antigen-antibody reaction of the labeling antigens or antibodies with fluorescein, radioisotopes, enzymes, or electron-dense substances [29]. This technology not only greatly improves the sensitivity of the antigen-antibody reaction and qualitatively or quantitatively analyzes trace substances, but also works in conjunction with microscopy or flow cytometry for positioning or quantitative analysis. For example, an enzyme-linked immunosorbent assay, anti-histone antibody, and anti-DNA antibody sandwich immunoassay are used to qualitatively and quantitatively analyze apoptosis according to the intensity of color reaction in the assays. The color products of the assays are formed after enzymatic catalysis when the nucleosomes are formed during cell apoptosis. In addition, flow cytometry can be used to detect apoptotic cells after Annexin V/propidine iodine (PI) staining. Annexin V is used as a fluorescent probe to label the exposed phosphatidylserine (PS) on the outer cell membrane. PI is a nucleic acid dye that cannot penetrate intact cell membranes. PI specifically binds to DNA. The amount of PI binding is positively correlated with the DNA content [30,31]. DNA can also be labeled with dyes to observe the cell cycle distribution. Ca²⁺ in the cells are labeled using Rhod-2, Indo-1, and other specific fluorescent probes and then qualitatively and quantitatively analyzed by flow cytometry [32,33]. A fluorescent Elisa is also used to assess the activities and content of caspases and Cyto-C in apoptotic cells [21]. Western blotting is another approach that can measure the expression of apoptotic proteins (e.g. proteins in the endoplasmic reticulum apoptosis pathway).

3.3. Advantages and disadvantages of different immunological techniques and their application scope

An Elisa is a highly sensitive, qualitative, and quantitative detection for apoptosis. However, over-lysis of the cells results in coloring of the DNA fragments within the nuclei. This approach cannot be used for localizing the apoptotic site in cells and tissues, but it is suitable for the detection of apoptosis at different stages and for the large-scale qualitative and quantitative detection of apoptosis [34,35].

Analysis of Annexin V- and PI-stained cells via flow cytometry can be used to distinguish and quantify the level of apoptotic cells during the early, middle, and late stages of apoptosis, as well as in necrosis. This detection method is more sensitive than TUNEL. However, to prevent cell membrane damage, a strict operation in flow cytometry is required, and this method is also expensive [36]. Measurement of the cell cycle reflects the relationship between cell cycle and apoptosis. A hypodiploid peak showing reduced DNA content before the peak at the G0/G1 phase in the DNA histogram from flow cytometry represents the peak of apoptotic cells. This peak is used to determine the occurrence of apoptosis. In addition, the cells with sub-G1 DNA to the total cell ratio represent the percentage of apoptosis. However, this detection method is affected by the reaction time and temperature, resulting in varying degrees of small DNA fragments and overlapping peaks of apoptotic and non-apoptotic cells. Therefore, it is suitable for the detection of the early stage of apoptosis [36].

The immunofluorescence labeling of Ca²⁺ is suitable for the detection of apoptosis mediated by the endoplasmic reticulum through the endoplasmic reticulum pathways. The significant changes in Ca²⁺ concentrations that occur as a result of endogenous endoplasmic reticulum stress in apoptotic cells are revealed by fluorescence microscopy [37]. Flow cytometric analysis of the DNA content after DNA is specifically bound to a fluorescent dye, such as PI, can also be used for the analysis of apoptosis. The amount of fluorescent dye bound is positively correlated with the DNA content. Thus, after PI binds to the DNA, the DNA can be analyzed after linear and logarithmic amplification. The DNA content in apoptotic cells is reduced due to endonuclease cleavage. During preparation of the specimen, the integrity of the cell membrane is destroyed, and the DNA fragments of the apoptotic cells flow out of the cells, which may also lead to a decrease in the overall DNA content [36].

An fluorescence ELISA is used to assess the activities and contents of caspases and Cyto-C and is suitable for evaluating the various inducers of apoptosis in vitro because of its high sensitivity, strong specificity, and no cross-reactivity with known caspases. The activity of caspases and Cyto-C decreases during the late stage of apoptosis [38,39]. A specific fluorogenic substrate for activated caspase-3, Ac-DEVD-AFC, is added to the cells to evaluate the activity of caspase-3 through assessing the levels of decomposition of the fluorescent cleavage product, AFC, or the level of the fluorescence intensity [40]. It is sometimes necessary to indirectly determine the degree of apoptosis by analyzing the ratio of cleaved-caspase-3 to pro-caspase-3. This method is not suitable for detection of the late stage of apoptosis.

Western blotting is a method that can be used to detect apoptosis by analyzing the changes in endogenous mitochondrial and endoplasmic reticulum stress unfolded-protein response pathways and the changes in the expression of proteins related to the apoptosis pathway of exogenous death receptors. Regardless of the apoptosis-related signaling pathways, this method does not reflect the morphological changes of apoptotic cells. It is suitable for the detection of the early, middle, and late stages of apoptosis.

4. Conclusions

There are many techniques available for the detection of apoptosis. This article reviewed the aspects and principles of relevant apoptosis-detection methods based on the morphological, biochemical, molecular biological, and immunological aspects of apoptosis, summarized the advantages and disadvantage of the individual methods, and clarified the scope of application for different research objectives. A combination of molecular biology and immunological techniques has confirmed that tissues and cells induce apoptosis by first activating exogenous death receptor signaling pathways or related targets of endogenous apoptosis pathways (i.e. mitochondrial pathways and endoplasmic reticulum stress pathway) both in vivo and in vitro. Apoptotic cells show specific morphological features, the DNA ladder, biochemical characteristics of DNA fragments in apoptosis, and the characteristics of immunological antigen-antibody enzyme-linked reactions (Figure 1).

With the development of technologies, increasing methods for apoptosis detection are available. Regardless of the development, the core of the technologies is mainly focused on 3 aspects: (1) Primary observation of apoptotic bodies and chromatins by morphological microscopy combined with immunofluorescence or immunocytochemical/immunohistochemical staining; (2) Detection of the DNA ladder and DNA fragmented ends by gel electrophoresis, TUNEL, and flow cytometry quantitative analysis; and (3) Detection of targets related to apoptosis-related signaling pathways based on molecular biological, immunological, and morphological methods combined with fluorescence enzyme immunoassay (Figure 1). Irrespective of the single use of various technologies or application of composite technologies, it is necessary to choose the detection method(s) best suited to the research objective. If the research is to localize the apoptotic cells, a TUNEL method or related immunofluorescence staining can be used. Flow cytometry can be used to quantify the apoptotic cells in vitro. In regards to the phase of apoptosis, mitochondrial membrane potential is suitable for the detection of the early stage of apoptosis [41], while agarose gel electrophoresis is suitable for the detection of the late stage of apoptosis [16], and in situ DNA nick end labeling is suitable for the detection of the middle and late stages of apoptosis [41] (Table 1).

Each method has its own scope of application and advantages and disadvantages. Morphological detection is intuitive. However, not all apoptotic cells have typical morphological features of apoptosis. Thus, morphological detection is only a qualitative method. Although biochemical characteristics of apoptotic cells are significant, their corresponding detection methods are only suitable for qualitative detection of small samples and fail to distinguish which types of cells or tissues undergo apoptosis [42]. Immunology-related methods are suitable for the detection of apoptosis in single cells and cannot accurately and absolutely quantify and localize the apoptotic cells. Molecular biology techniques can clarify the mechanism of how apoptosis occurs. However, apoptosis signaling pathways can be initiated separately. Additionally, crosstalk between signaling pathways may also occur, especially between the mitochondrial-mediated apoptosis pathway [43,44], the endoplasmic reticulum stress pathway [45,46], and the death receptor pathway [47,48], all of which have aroused the attention of scientists. There are many targets to be analyzed in the apoptosis pathways. In addition, the expensive reagents may limit the use of a particular apoptosis-detection method. Complicated operation procedures for the detection of apoptosis also have the risk of incurring experimental errors.

Therefore, it is our goal to select simple and efficient detection methods for apoptosis based on the research objective, technical principles, research funding, and available equipment. It is necessary to explore and develop detection methods for the early stage of apoptosis in order to help guide the selection of therapeutic drugs and treatment methods for some diseases.

Author contributions

All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding

This work was supported by the National Natural Science Foundation of China [NO. 81,774,071, NO. 81,704,056], Tianjin Municipal Natural Science Foundation [NO. 18JCYBJC94500,19JCYBJC26400].

Disclosure of potential conflicts of interest

No potential conflict of interest was reported by the author(s).