Assays for Apoptosis
One of the major problems in detecting apoptosis is that many features of apoptotic and necrotic processes overlap. Therefore it is frequently necessary to use several independent assays to draw definitive conclusions. Each of the currently used assays has its advantages and disadvantages that make it more suitable for some applications but not others. To choose the detection methods appropriate for your system, you must know the kinetics of apoptosis in your model because apoptosis in some cases may last for only 2-3 h and could be followed by necrotic cell death.
TUNEL (TdT-Mediated dUTP Nick-End Labeling) Detection Kits: DNA fragmentation is one of the hallmarks of apoptosis. It occurs in the later stages, usually following caspase activation. The fragmented DNA of apoptotic cells can be labeled with biotin- or FITC-dUTPs at 3'-OH DNA ends using terminal deoxynucleotidyl transferase (TdT). The cells with labeled DNA can then be visualized by light microscopy or quantitated by flow cytometry. The TUNEL method is sensitive and fast, but is subject to false positives from necrotic cells and cells undergoing DNA repair and division. To achieve reliable results TUNEL assay should be paired with another assay, for example, Annexin V detection. TUNEL-staining is usually more challenging in paraffin-embedded tissues due to protein cross-linking caused by fixatives. GenScript offers a series of TUNEL Apoptosis Detection Kits that are specifically designed to detect apoptosis in paraffin-embedded or frozen tissue sections and attached cells and use biotin or FITC-labeled nucleotides.
Annexin V-EGFP Kits: Apoptotic cells are known to display phosphatidylserine (PS) molecules on their outer membranes, which allows safe clearance of apoptotic debris without activation of inflammatory pathways. The timing of PS exposure may depend on the nature of the apoptosis-inducing agent. PS molecules can be detected using Annexin V, a Ca2+-dependent, phospholipid-binding protein with high affinity to PS. To better distinguish between apoptotic and necrotic cells, GenScript uses a second fluorescent dye, propidium iodide (PI), that only enters necrotic cells across damaged membranes. GenScript’s Annexin V assay is nonenzymatic, does not require fixation, and is suitable for both adherent and suspension cells. It uses annexin V fused with EGFP to detect PS by light microscopy or flow cytometry.
Caspase Activity Assay Kits: The caspases are a family of cysteine proteases that includes some of the main initiators and executors of the apoptotic process. They exist within the cell as inactive pro-forms or zymogens. These zymogens are cleaved to form active enzymes following the induction of apoptosis. The disadvantages of this method include the transient nature of caspase expression and the fact that caspase activation may not necessarily lead to apoptosis. GenScript offers a series of Caspase Colorimetric Assay Kits for assaying the activity of caspases 2, 3, 6, 8 and 9 using a spectrophotometer or a microtiter plate reader.
Mitochondrial Apoptosis Detection Kit (JC-1): The kit utilizes JC-1, a cationic dye that fluoresces differently in healthy cells and in apoptotic cells. In healthy cells, JC-1 accumulates and aggregates in the mitocondria, giving off a bright red fluorescence. In apoptotic cells, the altered mitochondrial transmembrane potential causes JC-1 to remain in the cytoplasm in its monomer form, fluorescing green. The fluorescent signals can be easily detected by fluorescence microscopy using a band-pass filter (detects FITC and rhodamine) or analyzed by flow cytometry using the FITC channel for green monomers (Ex/Em =488/530+ 30 nm) and the PI channel for red aggregates (Ex/Em =488/590+ 42 nm).
Cell Apoptosis DAPI Detection Kit: The one-color flow cytometry assay kit contains diamidino-2-phenylindole (DAPI)DNA-specific dye. This dye can pass through intact, living cell membranes, and uptake of DAPI, leaving a stronger blue stain. In addition, the nuclear morphology of normal cells is round, clear-edged, uniformly stained. Apoptotic cells show irregular edges around the nucleus, chromosome concentration in the nucleus, heavier coloring, and, with nuclear pyknosis, an increased number of nuclear body fragments. For these reasons, the intensity of the fluorescence can help researchers identify apoptotic samples.
Cell Apoptosis PI Detection Kit: The one-color flow cytometry assay kit includes includes ready-to-use propidium iodide (PI), a DNA-specific red-fluorescence dye (excitation/emission maxima ≈535/617 nm). Propidium iodide is only permeant to dead cells. Under fluorescent microscopy, normal cells are not stained, while dead cells show strong red fluorescence. The intensity of the fluorescence of apoptotic cells is between dead cells and normal cells. Under flow cytometry, DNA in the cells is degraded by endogenous nuclease activity and diffused out of cells as part of apoptosis. A highly definable sub-G1 peak occurs and is easily quantified by PI. Cells can then be sorted and further analyzed by microscopy to verify the presence of apoptotic cells. The change in DNA of the apoptotic cells is then assayed by flow cytometry.
Double Stain Apoptosis Detection Kit (Hoechst 33342/PI): The simple and rapid two-color flow cytometry assay that uses Hoechst 33342, a type of blue-fluorescence dye (excitation/emission maxima ≈350/461 nm when bound to DNA), stains the condensed chromatin in apoptotic cells more brightly than normal chromatin. Propidium iodide (PI), a red-fluorescence dye (excitation/emission maxima ≈535/617 nm when bound to DNA), is only permeant to dead cells. The staining pattern resulting from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic, and dead cell populations by flow cytometry and fluorescence microscopy.
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