Rather than providing a specific protocol, the following can serve as a general guide to the most frequently utilized options for apoptosis assessment. This is by no means a comprehensive list of options, as the field continues to evolve. For each assay listed, the cellular events and accompanying detection method are described. It may be useful to consider taking advantage of the multiparametric capability of flow cytometry by combining two or more of these assays of apoptosis in a given cell system. For example, Annexin V can be combined with a mitochondrial membrane potential probe rather than a viability dye. Or, multiple regulatory proteins can be combined to evaluate changes in expression with apoptosis induction. In addition to the items below, several review articles are listed as information sources.
Membrane Phosphotidyl Serine Exposure: In early apoptosis, the cell membrane loses the asymmetry of its membrane phospholipids and phosphotidylserine, a negatively charged phospholipid normally found in the inner leaflet of the plasma membrane becomes exposed on the cell surface. Exposed PS can be detected by binding with Annexin V (a calcium and phospholipid binding protein) conjugated with a fluorochrome. Combining detection using directly-conjugated Annexin V with viability exclusion using Propidium Iodide or 7-AAD (7-aminoactinomycin D) allows for assessment of non-apoptotic vs. early and late apoptotic cells. Numerous assay kits are commercially available.
Mitochondrial Membrane Permeability Transition: A frequently used apoptotic assay, mitochondrial membrane potential assays use permeant live cell probes. The dyes are retained in the mitochondria and thereby fluorescent while the transmembrane potential is intact and released when potential is disrupted (with corresponding loss in fluorescence). Among the probes available for MMP assessment are Rhodamine 123, CMX Rosamine, and TMRE. Choice of these or other similar dyes will depend upon experiment plan.
DNA Strand Breaks (TUNEL): Toward end-stage apoptosis, the final event of DNA strand breaks can be visualized using a TUNEL assay. Fragmented ends of DNA are accessible to terminal deoxynucleotidyl transferase (Tdt), which facilitates the incorporation of DNA precursors onto the fragmented ends. These DNA precursors are detected via antibody or are directly labeled with fluorochrome. There are several commercially available kits which utilize this principle.
Detection of Apoptotic Regulatory Proteins: Commercially available antibodies to some of the apoptotic regulatory proteins can be used in flow cytometry. Among these are the Bcl-2 family (Bcl-2, Bax, Bcl-XL) and the CD95 family of proteins. Antibodies are also available for the active subunit of Caspase 3 and cleaved poly ADP ribose polymerase (PARP). Utilizing more than one of these markers can help delineate subpopulations and apoptotic-stage related expression of both pro- and anti-apoptotic proteins.
Generation of Reactive Oxygen Intermediates: Reactive oxygen intermediates (ROI) play a host of biological roles in the cell-signaling, oxidative burst, etc. In apoptosis, the accumulation of ROI can induce stress leading to cell death. Detection of ROI involves the use of cell probes which are non-fluorescent in their inert state but are converted to fluorescent products upon oxidation by ROI present in cell. Examples of cell probes typically utilized in this procedure include Dichlorofluorescein diacetate, dihydroethidium and dihydrorhodamine 123.
Glutathione (GSH): A complement to the ROI assay, this is a measure of glutathione (antioxidant) levels decreased as a result of increased production of ROI. Assessment of glutathione levels can be assessed by cell probes which react with cellular GSH and become highly fluorescent.
- Assay controls: In order to assess that the kit/method you choose is working, it is recommended that known biological positive and negative controls are employed, i.e. a known apoptotic-inducing system and corresponding negative control. Also, independent assessment of test cell condition is useful in correlating the status of cells at the time of assay.
- Monolayer cell removal: Scraping can physically disrupt cells and cause artifact in data interpretation, so it is best to use a cell removal method that involves a gentler method of cell removal. This is especially a problem in assays such as Annexin V which usually also contain a membrane impermeant cell viability dye like propidium iodide.
- Monolayer cell removal, revisited: The dead/dying cells are usually loose and/or floating, therefore it is essential to retain these cells for analysis purposes.
- Assay Timing: For any given apoptosis-inducing system, obtaining optimal flow cytometry data will depend on the timing of the assay; therefore performing a treatment time course is recommended for any unknown system.
- Apoptosis Regulatory Protein Detection: As these intracellular proteins are all detected using antibodies following some type of fixation/permeabilization, all of the technical considerations listed under Intracellular Antigen Detection apply.
Steemsa DP, Timm M, Witzig TE. Flow cytometric methods for detection and quantification of apoptosis. Methods in Molecular Medicine, 85:323-32, 2003.
Omerod MG. Using flow cytometry to follow the apoptotic cascade. Redox Report, 6 (5):275-87, 2001.
Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. Journal of Immunological Methods, 243(1-2):167-90, 2000 Sep 21.
Gorczyca W, Melamed MR, Darzynkiewicz, Z. Analysis of apoptosis by flow cytometry. Methods in Molecular Biology, 91:217-38, 1998.
Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20, 1997.
Expected Results: Multiparameter Apoptosis Assessment
Expected Results: Apoptotic Regulatory Protein Detection
Expected Results: Sub-G1 DNA