Apoptosis

Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics. The early process of apoptosis is along with cell shrinkage and pyknosis. And the cells are smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Apoptosis is different from necrosis. Necrosis is along with cell swelling, disrupted organelle and cell membranes, cytoplasm released and recruitment of inflammatory cells. Whereas apoptosis leads to cell death with cell shrinkage, intact cell membrane, cytoplasm retained in apoptotic bodies and without inflammation.
There are three main apoptotic pathways: the extrinsic (death receptor mediated), the intrinsic (mitochondrial) pathway, and perforin/granzyme pathway. Specifically, extrinsic pathway initiates apoptosis through death receptors-mediated interactions, and activates caspase-8. Intrinsic pathway initiates apoptosis through non-receptor-mediated stimuli, such as radiation, toxins, hypoxia, hyperthermia, viral infections, and free radicals. it produces intracellular mitochondrial-initiated events (such as loss of the mitochondrial transmembrane potential) and activates caspase-9. In perforin/granzyme pathway, it involves T-cell mediated cytotoxicity, and can induce apoptosis via granzyme B or granzyme A. Granzyme B can activate caspase-10 or directly activate caspase-3, but granzyme A works in a caspase-independent way. Moreover, three pathways all lead to the same terminal pathway, which is initiated by the cleavage of caspase-3. And the activation of caspase-3 results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors^[1]。

1. TEM based on cytomorphological alterations

Transmission Electron Microscopy (TEM) is still considered as the gold standard to confirm apoptosis. The main characteristics in apoptosis are: (1) electron-dense nucleus (marginalization in the early phase); (2) nuclear fragmentation; (3) intact cell membrane; (4) disorganized cytoplasmic organelles; (5) large clear vacuoles; and (6) blebs at the cell surface. The main disadvantages of TEM are the cost, and can only assay a small region at a time, as well as unable to detect apoptotic cells at the earliest stages^[1][2]。

2. Annexin V staining

Annexin V belongs to the calcium-dependent phospholipid-binding proteins, and can adhere specifically to phosphatidyl serine (PS). In healthy cells, PS is located in the cytosolic side of the plasma membrane. But PS can translocate to the extracellular leaflet of the membrane in the early stage of apoptosis. Therefore, fluorescently labeled Annexin V (such as FITC, EGFP-labeled Annexin V) can be used to detect the presence of PS in the extracellular side of the cell membrane in early apoptosis. Propidium iodide (PI) can bind efficiently to DNA only in damaged cells due to their high DNA affinity, and is excluded by intact cells. So that PI can be used to detect necrotic cells. In most cases, PI is combined with Annexin V to distinguish between apoptotic and necrotic cells. Annexin V-FITC/PI Apoptosis Detection Kit (HY-K1073) and Annexin V-EGFP/PI Apoptosis Detection Kit (HY-K1074) are widely used apoptosis detection kits^[3].

Annexin V/PI staining protocol can refer as below^[4]。

1. For suspension cells: Centrifuge at 1000 g for 5 minutes and then discard the supernatant. Add 1 mL of pre-cooled PBS to resuspend the cells, centrifuge at 1000 g for 5 minutes and then discard the supernatant.

For adherent cells: Collect the cell culture medium. Wash cells with PBS and add trypsin (without EDTA) to dissociate cells. Add the medium and gently suspend the cells to make a single-cell suspension. Centrifuge at 1000 g for 5 minutes and then discard the supernatant. Add 1 mL of pre-cooled PBS to resuspend the cells, centrifuge at 1000 g for 5 minutes and then discard the supernatant.

2. Resuspend the cells in 195 μL Binding Buffer.

3. Add 10 μL DAPI, 10 μL Annexin V-FITC and 5 μL PI for 30 min at 4 °C in dark room.

4. Annexin V-FITC and PI fluorescence was immediately observed under confocal laser scanning microscope. Wavelength: Annexin V-FITC (Ex = 488 nm; Em = 525 nm, green), and PI (Ex = 550 nm; Em = 617 nm, red). Viable Cells are Annexin V/PI negative, early apoptotic cells are Annexin V⁺/PI⁻, whereas cells in late apoptotic stage are Annexin V⁺/PI⁺.

5. For quantitative determination of apoptosis, cell apoptosis after staining can also be assessed for by flow cytometry.

3. Mitochondrial membrane potential detection

Cell apoptosis has been considered to be associated with the opening of the mitochondrial permeability pores and loss of the electrochemical gradient. And the loss of mitochondrial transmembrane potential (ΔΨM) is a valuable indicator of apoptotic cells. JC-1 dye is a lipophilic, cationic dye that is widely used to detect ΔΨM change in multiple cell types. In healthy cells with relatively high ΔΨM, the JC-1 dye enters and accumulates in the mitochondria, forming J-aggregates (Ex/Em=585/590 nm, orange-red fluorescence). In apoptotic cells, the JC-1 dye enters the mitochondria to a lesser degree since the less negative ΔΨM, keeping J-monomer (Ex/Em=510/527 nm, green fluorescence). Therefore, the red/green fluorescence ratio of JC-1 can be used to assess the state of apoptosis, by using confocal laser scanning microscope or flow cytometry. JC-1 Mitochondrial Membrane Potential Assay Kit (HY-K0601) is a widely used ΔΨM detection kit^[5].

JC-1 staining protocol can refer as below^[6].

1. Culture cells in 6-, 12- , 24-, or 96-well plates at suitable density for special cells. Incubate the cells according to your experiment protocol.

2. Ensure that the JC-1 and DMSO has equilibrated to room temperature, and then prepare a 200 μM stock solution by dissolving the contents of one vial in DMSO provided.

3. For the control tube, allow the vial of CCCP has come to room temperature, add 1 μL of CCCP (50 mM). Incubate cells at 37°C for 5 minutes.

4. Add 10 μL JC-1 (200 μM) per well to make the final concentration of 2 μM. Incubate cells at 37°C, 5% CO₂ for 15-20 minutes.

5. For CLSM imaging: Wash three times with PBS gently, and add fresh medium without serum. Take images in the green and red fluorescence channel by CLSM imaging. The images were obtained at 488 nm excitation and 530 nm emission for green (JC-1 monomers) and at 543 nm excitation and 590 nm emission for red fluorescence (JC-1 aggregates).

For flow cytometry: After incubation, centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant. Wash cells twice with PBS and add 2 mL PBS to suspend cells and vortex to mix thoroughly. Centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant. Add 500 μL PBS to suspend cells. Analyze sample on a flow cytometer.

Representative images by using MCE JC-1 Mitochondrial Membrane Potential Assay Kit

4. Apoptosis-related biomarkers detection

As mentioned, apoptosis pathways all lead to the cleavage and activation of caspase-3. Thus, apoptosis process can be observed through the detection of caspase-3. Besides, other biomarkers of apoptosis include activated caspases 2/3/7/8/9, cytochrome c, Bcl-2/Bcl-xl/Mcl-1, PARP, etc. And these biomarkers can be detected in a variety of ways, including western blot, immunoprecipitation and immunohistochemistry^[8].

5. DNA fragmentation detection

In apoptotic cells, DNA is cleaved by an endonuclease, and DNA fragmentation occurs in the later phase of apoptosis, which results in a characteristic “DNA ladder” (when run on an agarose gel) with each band in the ladder separated in size by approximately 180 base pairs. Usually, the formed endonuclease cleavage products of apoptosis can be visualized either by DNA laddering technique or the TUNEL staining method (based on enzymatically end-labeling the DNA strand breaks). One Step TUNEL Apoptosis Detection Kit (FITC) (HY-K1078) and One Step TUNEL Apoptosis Detection Kit (Cyanine 3) (HY-K1079) are widely used TUNEL apoptosis detection kits^[1].