Flow cytometry fcm appears to be a choice technique for the accurate quantification of apoptosis and is a method which distinguishes apoptotic from nonapoptotic cells by means of dna staining. Measuring apoptosis using annexin v and flow cytometry. Apoptosis is the name given to a normal cell processthat of programmed cell death. Immunofluorescence can be used in combination with other, nonantibody methods of fluorescent staining, for example, use of dapi to label dna. Sun xm, macfarlane m, zhuang j, wolf bb, green dr, cohen gm. Label free cellbased assays using photonic crystal optical biosensors steven m. Enzymatic activation of procaspases results in generation of. Cells undergoing apoptosis display typical changes in their morphological and physical properties cell shrinkage, condensation of chromatin and cytoplasm which are well measurable by flow cytometry.
Apoptosis from ancient greek falling off is a form of programmed cell death that occurs in multicellular organisms. We thank dr junming guo, medicine institute of ningbo. Article information, pdf download for advances in cytochemical methods for. The method is based on the ability of tdt to label. Here, we found that ing5 overexpression increased autophagy, differentiation, and decreased proliferation, apoptosis, migration, invasion and lamellipodia formation in gastric cancer. Zenon alexa fluor 488 rabbit igg labeling kit molecular probes, cat.
Flow cytometry of jurkat cells treated with cccp to depolarize the mitochondrial membrane or staurosporine to induce apoptosis. Apoptosis, or programmed cell death, is a growthlimiting regulatory mechanism by which cells can trigger their own death in response to extracellular signals because of irreparable cellular or dna damage. The annexin v conjugate may be used in flow cytometry or in other in situ detection methods to facilitate the rapid and early detection of cell surface changes associated with apoptosis. These panels can assay multiple apoptosis markers at once. Learn about your options when you need an apoptosis assay or an apoptosis marker. Timing apoptosis assays apoptosis effects of apoptosis inducing agent different drugs or stimuli can have different effects on the same cell line or tissue.
Apoptosis assays apoptosis detection apoptosis markers. It is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. Detection of apoptosis using the bd annexin v fitc assay. Lower protein concentration levels of each apoptotic marker were detectable with the bioplex pro rbm apoptosis assays than with western blotting methods. For research use only, not for use in diagnostic procedures. By monitoring characteristic executioner caspasedependent events, such as caspase3 activity assays. Several microscope designs can be used for analysis. Evaluation of apoptosis imaging biomarkers in a genetic. The classical method to detect dna ladders is to examine fragmented genomic dna on an agarose gel. This assay uses a nuclear stain to identify cells and a tunel label to measure dna strand breaks. As the term implies, this is normally a well controlled and highly regulated series of events leading to the. Gaining insight into the techniques for detecting apoptotic cells will allow the. Cellmediated cytotoxicity assay by highthroughput direct cell counting in.
Apoptosis assays detection and methods by, prabhu thirusangu, research scholar, sahyadri science college, kuvempu university, shimoga 9th july,20 2. Detection of apoptosis using the bd annexin v fitc assay on the bd facsverse system bd biosciences august 2011 introduction apoptosis is a normal genetically programmed process that. Other mitochondrial dyes can be used that measure the redox potential or metabolic activity of the mitochondria in cells. A major drawback to these 51cr release assays is their reliance on the use of a. Apoptosis dna fragmentation analysis protocol a distinctive biochemical feature of apoptosis is the fragmentation of dna by a specific nuclease called caspaseactivated dnase cad. The pros and cons of apoptosis assays for use in the study. Furthermore, the methods used for the evaluation of caspase3 in vitro and in vivo were differentthe caspaseglo activity assay and ihc for cleaved caspase3, respectively. Markers of apoptosis in cardiovascular tissues cardiovascular. Advances in cytochemical methods for detection of apoptosis. Pdf applications of cytometry in necrobiology have two goals 1 to elucidate. A new label free method for the detection of apoptosis was proposed based on colorimetric assay of caspase3 activity using an unlabeled aspgluvalasp devdcontaining peptide substrate and.
We also offer tunel assays, apoptosis antibodies, inhibitors and other markers for apoptosis. Most of the apoptosis assays detect cellular changes late in apoptosis. It plays critical roles in development and immunity, as well as cancer and neurodegenerative disease. Cell death can occur by either of two distinct mechanisms, necrosis or programmed cell death apoptosis. Comparison of comet assay, electron microscopy, and flow.
Because dna fragmentation is one of the most reliable methods for detecting apoptosis, we have collaborated with phoenix flow systems to offer the apobrdu tunel assay kit, which provides all the materials necessary to label. Robust highthroughput kinetic analysis of apoptosis with realtime. We have experimental templates for most common apoptosis assays. Regardless of the method used to detect apoptosis, appropriate controls must be included, as experimental conditions and cell type can alter the performance of some assays. In situ apoptosis detection kit during apoptosis, dna is fragmented following the apoptotic activation of intracellular endonucleases. Necrosis is a pathological process which occurs when.
Colorimetric detection of apoptosis based on caspase3. Reagents to assess apoptosis, including tunel assays, annexin v, and an. Rapid onchip apoptosis assay on human carcinoma cells based on annexinvquantum dot probes. This semiquantitative method is a simple technique that provides a robust answer. Pdf applications of cytometry in necrobiology have two goals 1 to. Annexin v binding of cell surface phosphotidylserine. Another method to assess cell viability is dye exclusion, using an impermeant dye. Cunninghamb received 11th november 2010, accepted 4th january 2011 doi. The method is based on the ability of tdt to label blunt ends of doublestranded dna breaks independent of a template. Terminal deoxynucleotidyl transferase tdt dutp nickend labeling tunel assay has been designed to detect apoptotic cells that undergo extensive dna degradation during the late stages of apoptosis. Recognized by a variety of assays, activated caspases cleave many cellular proteins and the resulting fragments may serve as apoptosis markers. Apoptosis assays are wideranging, partly because the process itself can be divided into early, middle and late stages depending on which pathways are studied. Stepbystep protocols for apoptosis detection, such as use of the apoptotic cell surface marker annexin v assay, single strand dna antibody assays, caspase substrate assays.
Comet assay has been introduced to many different cell models as a. The latter strongly depends on techniques to measure apoptosis. Cell viability and death functional assay dna labeling assay morphological assay reproductive assay membrane integrity assay. Bioplex pro rbm apoptosis assays instruction manual for technical support, call your local biorad office, or in the u. Apoptosis and necrosis quantification kit a convenient assay for staining apoptotic cells with green fluorescence and necrotic cells with red fluorescence for quantitation within the same cell population. Regardless of which cytometric method is used to identify apoptosis, the. Hcs apoptosis tunel assay thermo fisher scientific us.
Some agents have no effect on a cell line, whereas other agents might induce necrosis, rather than apoptosis, or even induce different apoptosis. Several assays for detection of apoptosis were also developed during this past decade. Rapid onchip apoptosis assay on human carcinoma cells. Understanding if cell death is a result of apoptosis is important for understanding developmental pathways, the mechanism of action of small molecule and large molecule biologics drugs, and related functions of the immune system. Apoptosis testing assays can determine cell death, or apoptosis. Tunel assays are the most widely used method to detect apoptotic dna fragmentation in tissue samples and also work well for cultured cells. Apoptosis is the cellular process of programmed cell death. Learn about cell health assays and choose your ideal. Various methods available for detection of apoptotic cells. A new labelfree method for the detection of apoptosis was proposed based on colorimetric assay of caspase3 activity using an unlabeled aspgluvalasp devdcontaining peptide substrate and. Bioplex pro immunoglobulin isotyping microrna expression. Detection of apoptosis in vivo using antibodies against caspaseinduced neoepitopes sabine jakob, nadia corazza, eva diamantis, andreas kappeler, thomas brunner pages 255261. Detection of apoptosis by tunel assay springerlink.
Cooper jc, cassadycain rl, wooding c, linton k, alexander dr, higgins cf. The in situ apoptosis detection kit is designed to. Pdf cytometric methods to detect apoptosis researchgate. Bak and bclxl assays with that of western blotting figure 3. Pdf colorimetric detection of apoptosis based on caspase. Gaining insight into the techniques for detecting apoptotic cells will allow. The ability of tumor cells to elude apoptosis is a hallmark of most types of cancer. Cytotoxicity and apoptosis induction of some selected marine. The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.
Apoptosis detection and assay methods sigmaaldrich. The bestrecognized biochemical hallmarks of apoptosis are the activation of cysteine proteases caspases, condensation of chromatin, and fragmentation of genomic dna into nucleosomal fragments. However, these dyes do not address the mechanism of cell death and should be used in conjunction with other apoptosis detection methods such as a caspase assay. Phosphatidylserine exposure during apoptosis reflects. Here cytometric assays have been applied to quantify the expression of cell. The main part of this article will, however, focus on a recently introduced technique, the annexin vbased apoptosis detection assay. Ing5 suppresses proliferation, apoptosis, migration and. Apoptosis assays typically detect biochemical and cellular events that indicate activation of an apoptotic pathway. This book provides cuttingedge methods in apoptosis detection, and broad indepth coverage of current apoptosis research methodologies. Phosphatidylserine ps exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin v binding assay to detect. Analysis of apoptosis by cytometry using tunel assay. Comet assay has been introduced to many different cell models as a convenient. Extensive fragmentation of nuclear dna that generates a large number of dna double strand breaks is one of the most characteristic events of apoptosis 1,2. Detection of apoptosis by the tunel assay springerlink.
Annexin v kits biorad offers a quick and easy method for detecting apoptotic cells by flow cytometry using the annexin v apoptosis kits offered in a variety of sizes, ranging from a 20 or 50 test trial size. There are many different methods available, as well as commercial kits for this purpose. In other experiments, we employed a modification of a budr labeling method. Here we focus on five main types of apoptosis assay. Medial smooth muscle cell loss in arterial allografts occurs by cytolytic.