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Cell Apoptosis/Methods for the detection of apoptosis
Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Compared to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is a highly regulated process that determines a cell’s fate.
Figure 1. Degradation of lipid droplets by ATTECs
Figure 1. Intrinsic and Extrinsic pathways of apoptosis [1].
Many characteristic changes occur during the apoptosis process, including membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, formation of apoptotic bodies and finally phagocytosis.
Figure 2. The process of apoptosis
Figure 2. The process of apoptosis.
Detection of apoptosis

There are many methods to detect apoptosis which can roughly be divided into three categories: based on cell morphology, biological function and biochemical markers. Some of the most commonly employed assays are described below.

Cell Morphology Based Assays

1.1 Electron Microscopy Analysis

Electron microscopy analysis has always provided us with a great deal of data in cell apoptosis research. Transmission electron microscopy (TEM) is the most classic and reliable method for apoptosis so far, to distinguish between apoptotic and necrotic cell death based on distinct ultrastructural features as it offers high resolving power (0.1-0.4 nm).

As shown in Fig 3, TEM revealed that BBR (Berberine) or DDP (Cisplatin) induced apoptosis in significant number of OVCAR3 cells. Changes mainly include cytoplasmic shrinkage, blebbing of the plasma membrane, chromatin condensation and the formation of apoptotic bodies, while combination treatment of BBR and DDP induced apoptosis in relatively more no of cells.

Figure 2. In vitro reprogramming of B. subtilis ClpCP by BacPROTAC-1[5].
Figure 3. BBR and/or DDP induced apoptosis in OVCAR3 cells [2].
The gross changes and apoptotic morphology were detected by Transmission Electron Microscopy (TEM) assay under 4200× magnification.

1.2 Nuclear Staining Assay

In apoptotic cells, there is a change in plasma membrane permeability and chromatin condensation that can easily be detected with DAPI or Hoechst fluorescent dyes (Fig. 4). This is a quick and simple assay for quantification of apoptosis.

Figure 4. Fragmentation and nuclear condensation were observed by Hoechst 33342 staining
Figure 4. Fragmentation and nuclear condensation were observed by Hoechst 33342 staining[3].
PC-3 cells were treated with stem extract of Momordica cochinchinensis (0, 0.3, 0.6, 0.9, and 1.2 mg/mL) for 24 h. Treated cells exhibited morphological changes in the nuclei, typical of apoptosis. Photographs were taken under a fluorescence microscope (20×). Arrows represent apoptotic cells.
Biological Functions Based Assays

2.1 Mitochondrial Membrane Potential Assay

Apoptosis results in the increase of mitochondrial membrane permeability and loss of mitochondrial membrane potential (ΔΨm). Reduced mitochondrial membrane potential is considered as an early indicator and irreversible step towards apoptosis.

Lipophilic cationic dyes, like JC-1 (HY-K0601) (Fig. 5), have been widely used for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix and forms a polymer that emits strong red fluorescence (Ex/Em=585/590 nm). However, when the mitochondrial membrane potential is low, JC-1 cannot aggregate in mitochondrial matrix and produces green fluorescence (Ex/Em=510/527 nm).

Figure 5. Detection of mitochondrial membrane potential by JC-1 staining
Figure 5. Detection of mitochondrial membrane potential by JC-1 staining[4].
A549, H157, Calu-1 and H292 cells were treated with increasing concentrations of KRA-533 before the measurement.

2.2 Phosphatidylserine Assay

Phosphatidylserine (PS), a glycerophospholipid, is a critical component of the cellular plasma membrane. In normal live cells, PS is located on the cytoplasmic side of the cell membrane. Upon initiation of apoptosis, PS translocates from inner side to the outer leaflet of the membrane. Annexin V is a Ca2+-dependent phospholipid-binding protein. Annexin V can conjugate with fluorescence probes, like FITC, EGFP, PE, mCherry, etc, and is widely used as a sensitive indicator of early stage apoptosis cells due to its high affinity for PS. FITC conjugated Annexin V can emit green fluorescence. Propidium Iodide (PI) is a red fluorescent dye that cannot penetrate membrane of live cells or early apoptotic cells, but can stain late apoptotic cells and necrotic cells. After staining with Annexin V-FITC and PI, live cells show almost no fluorescence (Annexin V-/PI-), early apoptotic cells show green fluorescence (Annexin V+/PI-), and late apoptotic cells and necrotic cells show both green and red fluorescence (Annexin V+/PI+).

Figure 6. Type and Structures of selected molecular glue
Figure 6. SW480 cells were treated for 48h with PBR or Cetuximab, and were then stained with Annexin v-FITC/PI for flow cytometric analysis[5].
Biochemical Markers Based Assays

3.1 Apoptosis Marker Assays

Caspases (cysteinyl-aspartic acid proteases) are a family of cysteine proteases which play an essential role during apoptosis. Caspase-3 acts as an inactive pro-caspase in viable cells and becomes “activated executioner caspase” during apoptosis. Cleaved “poly-ADP-ribose polymerase 1” (PARP), a major substrate of executioner caspases, is usually used as a marker of apoptosis (Fig. 7).

Figure 7. ZnO-NPs Induced Caspase-Dependent Apoptosis in human Ca9-22 cells
Figure 7. ZnO-NPs induced caspase-dependent apoptosis in human Ca9-22 cells[6].

3.2 DNA fragmentation assay

In late stage of apoptosis, Caspases activate “Caspase-Activated DNase (CAD)`” that cleaves the DNA between nucleosomes into 180-200bp nucleosomal unit fragments which can be detected on agarose gel (Fig. 8).

Figure 8. DNA fragmentation assay
Figure 8. DNA fragmentation assay [7].
Lanes 1-4 show DNA fragmentation of cells treated with 0 (lane 1), 8.33 (lane 2), 333.33 (lane 3) and 666.67 pM (lane 4) of MCP30 in Ishikawa H cells for 72 h. DNA ladder was observed in lane 3 and lane 4.

3.3 In Situ DNA Fragmentation Assay (TUNEL Assay)

In late stage of apoptosis, Caspase-Activated DNase (CAD) not only cleaves DNA between nucleosomes, but also generates free 3’-OH groups at the ends of these DNA fragments. Fluorescent probe-labeled dUTP can be added to the exposed 3'-OH groups by “terminal deoxynucleotidyl transferase” (TdT) ,which is called TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling). TUNEL signal can be detected by fluorescence microscopy or flow cytometry (Fig. 9).

Figure 9. DNA fragmentation caused by hep 25. Cells were treated with various concentrations of hep 25, and the DNA damage was determined using the TUNEL assay
Figure 9. DNA fragmentation caused by hep 25. Cells were treated with various concentrations of hep 25, and the DNA damage was determined using the TUNEL assay[8].
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Apoptosis and Necrosis assay Kit

The staining of both Hoechst 33342 and propidium iodide (PI) was used to rapidly detect cell apoptosis and necrosis.

Cell Cycle and Apoptosis Analysis Kit (PI staining)

Cell cycle and apoptosis were detected by propidium iodide (PI) staining.

Cell Apoptosis Analysis Kit (Hoechst staining)

Apoptosis was rapidly detected by Hoechst 33258 staining.

Annexin V-FITC/PI Apoptosis Detection Kit

After staining with Annexin V-FITC and PI, healthy cells show almost no fluorescence (Annexin V-/PI-), early apoptotic cells show green fluorescence (Annexin V+/PI-), and late apoptotic cells and necrotic cells show both green and red fluorescence (Annexin V+/PI+).

Annexin V-EGFP/PI Apoptosis Detection Kit

After staining with Annexin V-EGFP and PI, healthy cells show no or low fluorescence (Annexin V-/PI-), early apoptotic cells show green fluorescence (Annexin V+/PI-), and late apoptotic cells and necrotic cells show both green and red fluorescence (Annexin V+/PI+). Compared with FITC, EGFP has stronger green fluorescence and more stable.

Mitochondrial Membrane Potential Assay Kit (JC-1)

JC-1 dye, a sensitive fluorescent probe, can be used as a quick and reliable indicator for the mitochondrial membrane potential of cells, tissues or purified mitochondria.

Please check our website for more information of the apoptosis kits!

All MCE products are only used for scientific research or drug registration application, we do not provide products and services for any personal or human use.

References
[1] Wu J, Ye J, Kong W, et al. Programmed cell death pathways in hearing loss: A review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 2020, 53(11): e12915.
[2] Liu L, Fan J, Ai G, et al. Berberine in combination with cisplatin induces necroptosis and apoptosis in ovarian cancer cells. Biol Res. 2019, 52(1): 37.
[3] Chainumnim S, Saenkham A, Dolsophon K, et al. Stem Extract from Momordica cochinchinensis Induces Apoptosis in Chemoresistant Human Prostate Cancer Cells (PC-3). Molecules 2022, 27(4): 1313.
[4] Xu K, Park D, Magis AT, et al. Small Molecule KRAS Agonist for Mutant KRAS Cancer Therapy. Mol Cancer. 2019, 18(1): 85.
[5] Park HJ, Park JB, Lee SJ, Song M. Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer. Int J Mol Sci. 2017 Aug 11;18(8):1746. doi: 10.3390/ijms18081746. PMID: 28800074; PMCID: PMC5578136.
[6] L SW, Lee CH, Lin MS, et al. ZnO Nanoparticles Induced Caspase-Dependent Apoptosis in Gingival Squamous Cell Carcinoma through Mitochondrial Dysfunction and p70S6K Signaling Pathway. Int J Mol Sci. 2020, 21(5): 1612.
[7] Gu HZ, Lin RR, Wang HC, et al. Effect of Momordica charantia protein on proliferation, apoptosis and the AKT signal transduction pathway in the human endometrial carcinoma Ishikawa H cell line in vitro. Oncol Lett. 2017, 13(5): 3032-3038.
[8] Chen RC, Lan CY. Human Antimicrobial Peptide Hepcidin 25-Induced Apoptosis in Candida albicans. Microorganisms. 2020 Apr 17;8(4):585.