To examine the causes of life, we must first have recourse to death," uttered Victor Frankenstein upon beginning his search for the source from "whence the principle of life proceeds" and ultimately creating his nameless monster. Frankenstein's real creator, 19-year-old Mary Shelley, probably had no idea when Frankenstein was first published in 1816 that her main character's motivation would eventually have real-life implications. Nearly 200 years later, the recourse to death provided by the investigation of programmed cell death, or apoptosis, has reached fruition with a handful of new drugs entering clinical trials. Designed to promote or inhibit apoptosis as required, these drugs are promising treatments for various cancers, stroke, and organ- transplant rejection.1
Apoptosis researchers have no shortage of tools available to aid them in their work.2,3 Among the many applications and methods, flow cytometry has proven to be particularly valuable in the study of apoptosis. The methodology itself has evolved into a sophisticated technology providing researchers with a vital tool for investigating the cellular mechanisms underlying most biological systems. Information on cellular structure or metabolic status, for example, is readily obtained on a cell-by-cell basis with this powerful technique. Here, after treatment with a variety of fluorescent probes, cells are interrogated individually by a highly focused laser beam as they flow along in a rapidly moving stream of liquid. Up to 10,000 cells per second can be monitored simultaneously for up to six different cellular characteristics.
|Courtesy of BioSource International|
Apoptosis detection strategies exploit numerous pathways.
This profile explores some of the oldest and newest flow cytometric products and kits available for studying apoptosis.
Among the many morphological and biochemical changes indicative of apoptosis, loss of the plasma membrane is one of the earliest. In apoptotic cells, the phospholipid phosphatidylserine (PS) translocates from the inner to the outer leaflet of the plasma membrane. Macrophages recognize PS exposed on the cell surface of lymphocytes during the apoptotic process. The recognition and phagocytosis of apoptotic cells and bodies protect organisms from inflammation caused by exposure to cellular compounds, a process that usually accompanies necrosis. The extent of the exposure of PS to the external cell environment can be used to distinguish apoptotic from nonapoptotic cells.5 This method relies on the calcium-dependent phospholipid binding protein Annexin V. An anticoagulant, Annexin V has a very high affinity for PS. When conjugated to a fluorochrome such as fluorescein isothiocyanate (FITC), Cy5, or phycoerythrin (PE), Annexin V retains its high affinity for PS, forming the basis for a sensitive flow cytometric assay for cells undergoing apoptosis. Furthermore, because the assay is nonenzymatic and does not require fixation of the cells, live apoptotic cells can be selected by fluorescence activated cell sorting (FACS).
FITC-Annexin V binding has become the hallmark method for distinguishing necrotic cells from those undergoing true apoptosis. This technique employs a double staining regime using a combination of Annexin V and a vital dye, such as the red fluorescent DNA-binding compound propidium iodide (PI). Because PI does not penetrate live cell membranes or early apoptotic cells, its presence, along with Annexin V, indicates cells that have lost membrane integrity as a result of necrosis or very late apoptosis. In comparison, early apoptotic cells are stained only with Annexin V.
Kit manufacturers and suppliers have developed a variety of apoptosis detection kits based on Annexin V binding. Annexin V coupled to FITC is the most popular approach, found in products such as the ApoDETECT™ Annexin V-FITC Kit from Zymed Laboratories of South San Francisco, Calif., the Annexin V-FITC Apoptosis Detection Kit from Oncogene Research Products of Boston, the Annexin V-FLUOS Staining kit from Roche Molecular Biochemicals of Indianapolis, and the Annexin V-FITC kit from StressGen Biotechnologies Corp. of Victoria, British Columbia. Sigma-RBI of St. Louis offers Annexin V-FITC and -Cy3 Apoptosis Detection Kits. Biotinylated Annexin V also appears in various products, such as the TACS Annexin V-Biotin Kit from Trevigen Inc. of Gaithersburg, Md., the Annexin V-Biotin Apoptosis Detection Kit from MBL International of Watertown, Mass., and the ApopNexin Biotin Apoptosis Detection Kit from Intergen Co. of Purchase, N.Y. The biotinylated Annexin V bound to the apoptotic cells can be conjugated with any avidin-bound fluorochrome.
Two Annexin V-based kits from Molecular Probes of Eugene, Ore., are built around Annexin V conjugated to Alexa Fluor™ 488. The Vybrant™ Apoptosis Assay Kit #1 is a single-color kit and the Vybrant Kit #2 is a two-color kit containing PI. Molecular Probes' Vybrant Apoptosis Assay Kit #6 uses biotinylated Annexin V that is detected with the blue fluorescent Alexa Fluor 350 conjugate of streptavidin and also uses PI. Using red and blue fluorescence leaves the rest of the visible spectrum available for multiparameter studies.
CLONTECH of Palo Alto, Calif., offers products containing Annexin V fused to enhanced green fluorescent protein (EGFP). Because there is a 1:1 ratio of EGFP to PS, the results obtained from assays using Annexin V-EGFP are quantitative. Extraordinarily bright and resistant to photobleaching, Annexin V-EGFP is also highly suitable for experiments that require high sensitivity, such as fluorescence microscopy.
Via-Probe, the ready-to-use solution of the nucleic acid dye 7-amino-actinomycin D (7-AAD) from BD PharMingen of San Diego, is an alternative to PI. Its chief advantage is its ability to be used with PE and FITC-labeled monoclonal antibodies with minimal overlap between the 7-AAD, PE, and FITC fluorescence emissions. Additionally, 7-AAD works well with Annexin V-PE; the two reagents are the heart of BD PharMingen's Annexin V-PE Apoptosis Kit.
DNA fragmentation occurs as one of the final stages of cell death. During this process, endonucleases degrade the higher-order chromatin structure into fragments ranging from 50 to about 300 kb long. Subsequently, these fragments are further cleaved into pieces between 180 and 200 bases in length or smaller. All of this cleavage generates DNA fragments with free 3' hydroxyl ends, forming the basis of an apoptosis detection method called "TUNEL," or terminal deoxynucleotidyl transferase (TdT) -dUTP nick- end labeling.6 TUNEL relies on TdT to catalyze the template-independent addition of dUTP to the 3'-hydroxyl termini of double- and single-stranded DNA. The conjugation of the dUTP to biotin or various fluorescent reporter molecules enables the detection and quantitation of DNA fragmentation by flow cytometry.
Most of the available TUNEL assay kits employ dUTP conjugated to FITC to directly incorporate excitable bases into DNA fragments. This procedure eliminates the need for secondary detection steps, resulting in rapid, specific, and sensitive staining. Representative of the direct labeling products on the market are the APO-DIRECT™ Kit from Phoenix Flow Systems of San Diego (sold by Alexis Corp. of San Diego, BD PharMingen, and Chemicon International Inc. of Temecula, Calif.); the ApoAlert DNA Fragmentation Assay kit from CLONTECH; and MBL International's MEBSTAIN Apoptosis Kit Direct.
TUNEL assay kits using a two-step labeling strategy are based upon the incorporation of either biotinylated or brominated dUTPs (Br-dUTPs) into the DNA fragments. MBL International's MEBSTAIN Apoptosis Kit II (also distributed in the United States by Beckman Coulter Inc. of Fullerton, Calif., as the Apoptosis MEBSTAIN Kit) and the TUNEL Apoptosis Detection Kit from Upstate Biotechnology of Waltham, Mass., use the biotinylated dUTP strategy. One advantage of this strategy is the use of different fluorescent color schemes produced by selecting appropriate fluorescent avidin or strept-avidin conjugates. Kits that use Br-dUTP incorporation are manufactured by Phoenix Flow Systems and distributed by Alexis, BioSource International, BD PharMingen, Sigma-RBI, and Chemicon. In these kits, the sites labeled with Br-dUTP are identified with a labeled anti-BrdU monoclonal antibody directly labeled with FITC or PE or indirectly labeled with a biotin/streptavidin fluorochrome conjugate. According to Phoenix Flow Systems, recent evidence shows that Br-dUTP is more readily incorporated into the genome of apoptotic cells than nucleotides conjugated to larger ligands like fluorescein or biotin. The overall result is greater fluorescent signals when Br-dUTP labeled cells are analyzed by flow cytometry or fluorescence microscopy.
Many of these kits contain materials for positive and negative controls. For example, the APO-BRDU Assay from Phoenix Flow Systems contains positive and negative control cells already fixed for analysis by flow cytometry or microscopy. Trevigen's FlowTACS Apoptosis Detection Kit (also available from R&D Systems of Minneapolis) includes a reagent called TACS-Nuclease that allows researchers to generate sample-specific positive controls.
TUNEL kits, of course, have additional applications with techniques other than flow cytometry, such as fluorescence microscopy. To illustrate, Oncogene Research Products' Fluorescein-FragEL™ DNA Fragmentation Detection Kit detects DNA fragmentation while allowing simultaneous examination of morphological changes in cells histologically. In fact, the kit contains a special mounting medium designed to enhance and preserve fluorescein emission. Intergen's ApopTag® In Situ Apoptosis Detection Kit provides a sensitive method of detecting DNA fragmentation at the single-cell level using the TUNEL method. Peroxidase, fluorescein, and rhodamine conjugates can be visualized by flow cytometry or microscopy.
Some companies have bypassed the TUNEL route altogether, taking different paths to detection of nucleic acid changes in apoptotic cells. Molecular Probes' Vybrant Apoptosis Assay Kit #5, for instance, is a rapid, two-color flow cytometry assay that uses a combination of Hoechst 33342 and PI to detect the condensed state of chromatin in apoptotic cells. In this case, the Hoechst dye stains the condensed chromatin of apoptotic cells blue while the red fluorescent membrane-impermeable PI stains only necrotic cells with permeable membranes. The assay has been optimized for Jurkat cells; some modifications may be necessary for other cell types.
The Apoptosis/ssDNA Detection Kit, -IHC from Kamiya Biomedical of Seattle, detects apoptotic cells with a monoclonal antibody to single-stranded DNA. Developed for the sensitive immunohistochemical detection of apoptotic cells in suspensions and tissue sections, the Apoptosis/ssDNA Detection Kit also has applications in analyzing cell suspensions by flow cytometry. Overall, the kit exploits the decreased stability of apo-ptotic DNA to thermal denaturation caused by the proteolysis of DNA-bound proteins during the early phases of apo-ptosis. A highly specific monoclonal antibody to single-stranded DNA then detects the apoptotic DNA in its single-stranded conformation.7
The Delta Psi Fraternity
Disruption of mitochondrial transmembrane potential is one of the earliest events after apoptosis induction.8 Normally, cellular energy generated by mitochondrial respiration accumulates in the transmembrane space as an electron gradient called the mitochondrial transmembrane potential or *m. Disruption of *m occurs following the onset of apoptosis. Trevigen's DePsipher™ Kit for the Detection of Mitochondrial Membrane Potential *m uses a fluorescent lipophilic cation called DePsipher as a mitochondrial activity marker. DePsipher fluoresces differently in healthy cells than it does in apoptotic cells. In healthy cells, the dye accumulates and aggregates in the mitochondria, producing a bright red fluorescence. In apoptotic cells, DePsipher cannot aggregate in the mitochondria because of the altered transmembrane potential; therefore, it stays in the cytoplasm in its monomeric form, producing a green fluorescence. These fluorescent signals are analyzed by flow cytometry using the FITC channel for green monomers and the PI channel for red aggregates. Additionally, apoptotic and healthy cells can be viewed simultaneously by fluorescence microscopy using a wide band-pass filter. The MitoCapture™ Mitochondrial Apoptosis Detection Kit from BioVision of Palo Alto, Calif., and CLONTECH's ApoAlert® Mitochondrial Membrane Sensor Kit use similar technology to detect the disruption of *.
CLONTECH has developed another kit designed to assay independent changes in the mitochondrial and plasma membranes through flow cytometry. The ApoAlert® Apo 2.7/Annexin V-EGFP Kit detects the Apo 2.7 antigen, a 38 kDa protein localized on the mitochondrial membrane on apoptotic cells, with a PE-conjugated Apo 2.7 antibody. In addition, the kit uses Annexin V-EGFP to detect PS translocation in the plasma membrane.
Caspases, a family of 14 cysteine aspartyl proteases, play a crucial role in apoptosis. The tremendous number of products developed to study caspases and their substrates is testimony to their importance and is beyond the scope of this article. However, a few flow cytometry-based caspase products are now available and are profiled here.
Caspase-3 is a key protease that becomes activated during the early stages of apoptosis. Originally synthesized as an inactive proenzyme, the activated form cleaves and activates other caspases, in addition to cleaving specific targets in the cytoplasm and nucleus. Once activated, caspase-3 is a marker for cells undergoing apoptosis. BD PharMingen's Active Caspase-3 Apoptosis Kit incorporates a PE- conjugated polyclonal rabbit anti-active caspase-3 antibody. This kit contains the necessary reagents to measure apoptosis by flow cytometry based on the presence of active caspase-3. Additionally, biotinylated and FITC-conjugated forms of a rabbit anti-active caspase-3 monoclonal antibody are available.
Augmenting its PhiPhiLux class of fluorogenic substrates for caspase-3 and caspase-3-like activity, OncoImmunin Inc. of Gaithersburg, Md., recently has begun offering a new line of cell-permeable fluorogenic caspase substrates, known collectively as CaspaLux substrates. The cell-permeable nature of both classes of substrates enables the visualization of intracellular protease activities by multiparameter flow cytometry or standard fluorescence microscopy in living cells. The CaspaLux substrates, designed for caspase-1, caspase-6, caspase-8 (the caspase-3 processing enzyme), and caspase-9, detect early events in the apo-ptotic pathway before DNA degradation has started, according to OncoImmunin's Beverly Packard. Recently, the CaspaLux substrates have been used to demonstrate that the pattern of caspase activation is not only dependent on the apoptosis-inducing agent employed, but also on cell type.9
BioSource International has recently introduced the FITC-PARP Cleavage Site Specific Antibody. PARP, or poly (ADP-ribose) polymerase, is a DNA repair enzyme cleaved by caspases early in the apoptotic process. The FITC-PARP antibody recognizes the cleavage site in PARP after it has been exposed by caspase activity. Suitable for flow cytometry, immunohistochemistry, and western blotting applications, the FITC-PARP antibody functions well with adherent and suspension cells. BioSource has demonstrated the specificity of the FITC-PARP Cleavage Site Specific Antibody by blocking its staining of apoptotic cells with a synthetic PARP peptide. Roche Molecular Biochemicals offers another site-specific antibody called M30 CytoDEATH, Fluorescein. M30 CytoDEATH is a monoclonal antibody that recognizes a specific caspase cleavage site within cytokeratin 18. This site is not detectable within native cytokeratin 18 in normal cells. As a result, M30 CytoDEATH represents a unique tool for determining very early apoptosis in single cells and tissue sections by flow cytometry and immunocytochemistry.
Finally, the cell-permeable, irreversible inhibitor of caspase activity Z-VAD-FMK has been modified for use in flow cytometry and other applications for cells in culture. Intergen's CaspaTag™ Fluorescein Caspase Activity Kit includes a carboxyfluorescein derivative of Z-VAD-FMK for the general caspase detection kit called FAM-VAD-FMK. The kit detects active caspases in live cells and is suitable for use with flow cytometry, fluorescence microscopy, and fluorescence spectroscopy. CaspaTag kits detecting caspase-1 (YVAD), caspase-3 (DEVD), caspase-6 (VEID), caspase-8 (LETD), and caspase-9 (LEHD) are now available as well. Similarly, the CaspACE™ FITC-VAD-FMK In Situ Marker for Apoptosis from Promega Corp. of Madison, Wis., monitors caspase activity in situ and has applications for high-throughput screening procedures.
With these and other apoptosis products sure to come, who knows what the continuing study of cell death will illuminate about life?
Michael D. Brush (firstname.lastname@example.org) is a freelance writer in Orange, Calif.
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9. A. Komoriya et al., "Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates," Journal of Experimental Medicine, 191:1819-28, June 5, 2000.