Apoptosis detection in single cells via flow cytometry

  • Conclusive identification of phases of apoptosis
  • Flow cytometry to facilitate multiparameter apoptosis detection
  • Reproducible apoptotic protein analysis using recombinant antibodies

Apoptosis, a form of programmed cell death, occurs in multicellular organisms. To date, most of the key processes in cellular apoptosis regulation are identified and can be targeted by therapeutic strategies. The identification of the major regulators of apoptosis has boosted intense research efforts in developing therapeutic approaches that intervene with cell death, either in a pro- or anti-apoptotic direction.

Apoptosis can be divided into four major phases: initiation, early apoptosis, intermediate apoptosis, late apoptosis. Flow cytometry is widely used to analyze apoptosis due to the versatility afforded by its several technological benefits:

  • Multiparameter analytical capabilities – Ability to analyze several parameters of apoptosis in a single assay
  • High-throughput analysis of large numbers of cells
  • Quantitative data gathering on percentages, as well as absolute number of cells
  • Single cell resolution – individual cell-based mode of analysis

Although it is difficult to measure intrinsic apoptosis initiation by flow cytometry, the extrinsic pathway is easily measured by monitoring expression of death receptors (DR), which mainly act as a ligand upon binding, resulting in initiation of apoptosis. Two models have been suggested for extrinsic apoptosis initiation: TNF (tumor necrosis factor)-induced and the Fas-ligand mediated model.  TNF-induced apoptosis can be measured by monitoring CD120a (TNFR1), CD120b (TNFR2), CD261 (DR4/TRAILR1), CD262 (DR5/TRAILR2), and CD266 expression. Fas-ligand mediated apoptosis can be measured by monitoring CD95 (FAS) and CD178 (FASL) expression. 

Apoptosis of primary tumor cells and virally-infected cells can be induced by cytotoxic T lymphocytes and NK cells. Such apoptosis induction results from the release of the cytotoxic molecules Perforin, Granzyme A, and Granzyme B by the effector cell, which themselves can be measured by flow cytometry. 

The early phase of intrinsically mediated apoptosis can be measured by monitoring B-cell lymphoma 2 (Bcl-2) protein expression. The Bcl-2 family regulates apoptosis through the actions of both the pro-apoptotic and anti-apoptotic members. 

Extrinsic-pathway induced apoptosis is characterized by translocation of phosphatidylserine (PS) to the outer leaf of the plasma membrane, loss of mitochondrial membrane potential, cytochrome C ad ATP release, and by activation of caspase-8 and -9 during the early phase. 

In most normal, viable eukaryotic cells, the negatively charged phospholipid PS is located in the cytosolic leaflet of the plasma membrane lipid bilayer. PS redistribution from the inner to the outer leaflet is an early and widespread event during apoptosis. However, in necrosis, PS becomes accessible due to the disruption of membrane integrity. PS exposure by apoptotic cells serves as a trigger for recognition and removal by macrophages. Annexins are a family of structurally related proteins that can bind specifically to cellular membranes. Annexin V has a very high affinity for membranes containing the negatively charged phospholipid phosphatidylserine, and can be used as a marker of early apoptosis events. 

Analysis of early apoptotic and dead cells

Jurkat cells, cultured with staurosporine (50 nM) for 15 hours, were stained with Annexin V conjugates followed by staining with DAPI and analyzed by flow cytometry using the MACSQuant® Analyzer.

The intermediate stage of apoptosis is characterized by cytochrome c release, activation of caspase-3, 6, and 7, Poly-ADP-Ribose polymerase (PARP) cleavage, cell shrinkage, and activation of nucleases. 

Cytochrome c protein can be found in the inner membrane of the mitochondria and is an essential component of the electron transport chain. In apoptotic conditions, cytochrome c detaches from the mitochondrial inner membrane and migrates to the cytoplasm through pores in the outer membrane. Once released, cytochrome c activates caspase-9, which then in turn activates effector caspases. 

Analysis of Cytochrome c expression in human lymphocytes

Human lymphocytes were fixed and permeabilized followed by intracellular staining with Anti-Cytochrome c antibodies. Flow cytometry was performed using the MACSQuant Analyzer. Cell debris was excluded from the analysis based on scatter signals.

The late stage of apoptosis is characterized by DNA fragmentation, nuclear collapse, formation of apoptotic bodies, and phagocytosis by macrophages.

DNA fragmentation can be visualized by flow cytometry using DNA-binding dyes, such as PI, 7-AAD, DAPI, and Hoechst 33342. Cells in the late phase of apoptosis can be fixed and permeabilized using 70% ethanol. Such permeabilization results in the leakage of DNA fragments created during the late phase. Therefore these cells will have less DNA, and when stained with a DNA-binding dye, will exhibit less fluorescence than is normal. When represented on a DNA profile plot, which is usually represented on a linear scale, these cells appear as a sub-G1 population.

Another way to measure late apoptosis by flow cytometry is to monitor phosphorylation of histone variant H2A.X, which results in double-stranded DNA breaks. Phosphorylation of H2A.X at Ser139 correlates well with each double-stranded DNA break, phospho-H2A.X is a sensitive marker used to examine DNA damage and late apoptosis. 

Analysis of H2A.X phosphorylation in HeLa cells

HeLa cells were fixed and permeabilized using the Cell Signaling Buffer Set A. Cells were then stained with Anti-H2AX pS139 antibodies and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris were excluded from the analysis based on scatter signals.

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