Report CopyRight/DMCA Form For : Apoptosis Cell Cycle And Cell Proliferation
Cell proliferation is an increase in the number of cells as a result of growth and division Cell proliferation is apoptosis cell cycle and cell proliferation
Life Death and Cell Proliferation, The balance of cell proliferation and apoptosis is important for both development and normal. tissue homeostasis Cell proliferation is an increase in the number of cells as a result of growth. and division Cell proliferation is regulated by the cell cycle which is divided into a series of. phases Apoptosis or programmed cell death results in controlled self destruction. Several methods have been developed to assess apoptosis cell cycle and cell proliferation. BD Biosciences offers a complete portfolio of reagents and tools to allow exploration of the. cellular features of these processes, Over the years multicolor flow cytometry has become essential in the study of apoptosis. cell cycle and cell proliferation Success of the technology results from its ability to monitor. these processes along with other cellular events such as protein phosphorylation or cytokine. secretion within heterogeneous cell populations BD Biosciences continues to innovate in. this area with new products such as the BD Horizon violet cell proliferation dye 450. VPD450 and popular reagents such as antibodies to cleaved PARP and caspase 3 available. in new formats and for different types of applications. In addition to flow cytometry products BD Biosciences carries a broad portfolio of. reagents for determination and detection of apoptotic and proliferative events by ELISA. immunohistochemistry cell imaging or Western blot, As part of our commitment to maximize scientific results BD Biosciences provides a variety. of tools to assist customers in their experimental setup and analysis These include a decision. tree to guide in the selection of the most suitable methods for a specific study. BD Biosciences carries high quality reagents in the latest. formats to examine cell cycle proliferation and apoptosis. across a variety of platforms in applications from basic. research to drug screening, For Research Use Only Not for use in diagnostic or therapeutic procedures 3. Fundamental cellular processes,Cell Cycle and Cell Proliferation. An Overview, To help researchers better understand the fundamental The Cell Cycle. cellular mechanisms involved in immunity inflammation The cell cycle has two major phases interphase the phase. hematopoiesis neoplasia and other biological responses between mitotic events and the mitotic phase where the. BD Biosciences offers a range of tools including antibodies mother cell divides into two genetically identical daughter. kits and systems to measure proliferative responses Using cells Interphase has three distinct successive stages. flow cytometry immunofluorescence or immunohisto During the first stage called G1 cells monitor their. chemistry researchers can quickly and accurately determine environment and when the requisite signals are received. the cell cycle status or tissue localization of individual cells the cells synthesize RNA and proteins to induce growth. within proliferating populations These tools include When conditions are right cells enter the S stage of the cell. cycle and commit to DNA synthesis and replicate their. DNA dyes propidium iodide PI 7 aminoactinomycin D, chromosomal DNA Finally in the G2 phase cells continue to. grow and prepare for mitosis,Antibodies against cyclins retinoblastoma and. phosphorylated histone H3 M, In adaptive immunity specific T and B lymphocytes undergo. clonal expansion division proliferation and differentiation. in response to foreign antigenic stimulation Cell growth. replication and division in eukaryotic cells occur according Preparation. repara Growth, to a highly controlled series of events called the cell cycle 1 G2 Mitos. Mitosis aratio,Preparationn,Growth thesis,terp h ase. Replication,Cell cycle phases, Measures Reagents Mechanism Technology Sample Types. DNA Propidium Iodide PI 7 aminoactino Interaction into DNA double strands Flow cytometry Fixed permeabilized and for live dead. mycin D 7 AAD discrimination in intact cells, Cell Dyes BD Horizon violet proliferation dye 450 Diffuses into live cells and is hydrolyzed Flow cytometry Live proliferating cells. VPD450 by intracellular non specific esterases,to become fluorescent products. Newly Synthesized DNA BrdU and antibodies to BrdU Bromodeoxy uridine replaces thymidine Flow cytometry cell imaging Fixed and permeabilized cells treated. T in dividing DNA It is then detected immunohistochemistry tissues cell imaging immunohisto. by antibodies to BrdU chemistry only, Protein Level Antibodies to Ki67 PCNA Levels increase as a result of Flow cytometry bioimaging Fixed cells tissues and extracts. proliferation immunohistochemistry Western blot, Protein Level Antibodies to cyclins retinoblastoma Levels go up and down at different Flow cytometry bioimaging Fixed cells tissues and extracts. Rb other cell cycle markers stages of the cell cycle immunohistochemistry Western blot. Protein Modification Antibodies to phosphorylated histone Proteins become phosphorylated as Flow cytometry bioimaging. H3 cyclin dependent kinases cdk a result of proliferation or changes to immunohistochemistry Western blot. the cell cycle, BD CBA for quantitative detection Fixed cells tissues and extracts. Methods for the study of cell cycle and proliferation. 4 For Research Use Only Not for use in diagnostic or therapeutic procedures. CELL CYCLE,Relative Cell Number,Analysis of Cellular DNA Content. BD Biosciences offers a wide variety of reagents to study 70. the cell cycle Reagents include DNA dyes such as propidium. iodide PI and 7 amino actinomycin D 7 AAD In addition 0 200 400 600 800 1000. the BD Cycletest Plus reagent kit includes PI and other PI FL3 H. reagents to degrade proteins and RNA to allow more. precise DNA measurement The samples are subsequently B. analyzed using flow cytometry to assess ploidy identify. Relative Cell Number, abnormal DNA stemlines and estimate the DNA index DI 160. and cell cycle phase distributions of stemlines, During the cell cycle phases DNA levels change facilitating. the use of DNA dyes such as 7 AAD to generate characteristic 80. cellular DNA content profiles see the figure below. As cells go through the phases of the cell cycle proteins. such as histone H3 Ser28 become modified or change. in expression 2 To facilitate DNA replication the histone 0 200 400 600 800 1000. is modified opening the chromatin to allow entry of PI FL3 H. replication machinery To further support the study of cell. Measurement of DNA content using PI Two, cycle BD Biosciences carries antibodies to these proteins to mouse T cell lines MGG3 A and C20 4 B. use for imaging or flow cytometry applications were treated with 100 g mL RNase A and. then stained with 10 g mL of PI,Cell cycle analysis of a population. stained for incorporated BrdU and R4,total DNA levels 7 AAD. Human PBMCs were stimulated with,anti CD3 CD28 for 48 hours and. re stimulated with PMA ionomycin R6,for 4 hours and BrdU was added. for the final 1 hour Cells were then R6,harvested and stained using the. BrdU staining protocol,50 100 150 200 250 50 100 150 200 250. 7 AAD FL3 7 AAD FL3, For Research Use Only Not for use in diagnostic or therapeutic procedures 5. New tool to determine cell divisions,Tools and Techniques to Study. Cell Proliferation, Cell proliferation can occur in response to many stimuli VPD450 is a nonfluorescent esterified dye The ester group. such as cytokine exposure or a variety of other processes allows the dye to enter the cell Once the dye is inside the. BD has a new product to help researchers study cell cell esterases cleave off the ester group to convert the. proliferation dye into a fluorescent product and trap it inside the cell. With each replication event the amount of dye in the cell is. BD Biosciences offers Violet Proliferation Dye 450 for. decreased leading to a characteristic pattern, the detection of cell proliferation with the violet laser. which facilitates the use of larger panels This allows the. determination of more data from limited samples using. multicolor flow cytometry, The use of VPD450 to correlate cell proliferation with IL 2. production, CD4 enriched mouse splenocytes were loaded with 1 M VPD450. for 10 minutes Cells were then stimulated with anti CD3 CD28. and harvested at the indicated times Approximately 4 to 6 hours. prior to harvest cells were stimulated with PMA ionomycin in the. presence of BD GolgiStop protein transport inhibitor Cells were. fixed and permeabilized stained for IL 2 and analyzed on a. BD LSR II flow cytometer,Day 1 Day 2 Day 3,IL 2 Alexa Fluor 488. IL 2 Alexa Fluor 488,IL 2 Alexa Fluor 488,102 103 104 105 102 103 104 105 102 103 104 105. VPD450 VPD450 VPD450, 6 For Research Use Only Not for use in diagnostic or therapeutic procedures. P R O L I F E R AT I O N, Tools for BrdU Analysis In addition to DNA increases levels of certain proteins also. BD Biosciences carries a series of antibodies and kits rise as a result of cell proliferation For example Ki67 is an. designed for the detection of proliferating cells by antigen that is expressed in the nucleus of dividing cells. measurement of bromodeoxyuridine BrdU an analog However during the G0 phase of the cell cycle it is not. of the DNA precursor thymidine used to measure de novo detected Ki67 can be combined with other proliferation. DNA synthesis During the S phase of the cell cycle DNA markers such as BrdU and VPD450 for added confidence. synthesis BrdU is incorporated into the newly synthesized These markers can also be combined with cell surface and. DNA and can be readily detected by anti BrdU specific other types of markers to gain additional information about. antibodies BD antibodies and kits designed for the cell subsets and their signaling pathways. detection of BrdU are available for both intracellular. flow cytometry and immunohistochemistry and include. BD Horizon V450 and PerCP Cy 5 5 formats,1 g Aphidicolin. Cell cycle analysis of HeLa cells treated with Aphidicolin DNA. polymerase inhibitor monitored by BrdU staining The images. were captured on a BD Pathway 855 bioimaging system with. a 20x objective and merged using BD Attovision software. Hoechst Blue,Histone H3 pS28 Yellow,Tubulin Green, For Research Use Only Not for use in diagnostic or therapeutic procedures 7. The importance of tissue homeostasis,Techniques to Study Apoptosis. Programmed Cell Death, As cells become damaged or are no longer needed they However some cell types do not display characteristic. undergo apoptosis or programmed cell death a normal features of apoptosis In those cases multiple aspects. physiological process that occurs during embryonic of apoptosis might need to be analyzed to confirm the. development and tissue homeostasis maintenance mechanism of cell death. Apoptosis is an organized process that signals cells to To support this spectrum of requirements BD Biosciences. self destruct for cell renewal or to control aberrant cell offers a full range of apoptosis detection tools and. growth Apoptosis controls the orderly death of damaged technologies for measuring indicators at different stages. cells whereas necrosis occurs as a result of tissue damage across the apoptotic process BD Biosciences tools use multiple. causing the loss of both damaged and surrounding cells 3 methodologies including flow cytometry bioimaging and. microscopy for live and fixed cell analysis as well as ELISA. The apoptotic process is characterized by certain morpho. IHC Western blot and spectrofluorometry, logical features These include changes in the plasma. membrane such as loss of membrane symmetry and loss. of membrane attachment a condensation of the cytoplasm. and nucleus protein cleavage and internucleosomal, cleavage of DNA In the final stages of the process dying. cells become fragmented into apoptotic bodies and, consequently are eliminated by phagocytic cells without. Apoptotic Cells, significant inflammatory damage to surrounding cells. Intact Cells Cell Extracts Tissue Sections,Flow Cytometry Bioimaging Microscopy ELISA IHC. Live Fixed Live Fixed V i,Loss of Membrane Cleaved Markers i 6 V i. i Western Blot or,Asymmetry U V i,i i Immunoprecipitation. Mitochondrial TUNEL DNA Apoptosis Markers,i L i i Fragmentation. Spectrofluorometry,Caspase Activity, 8 For Research Use Only Not for use in diagnostic or therapeutic procedures. C E L L D E AT H,Annexin V PE Conjugate,Plasma Membrane Phospholipid Flipping. Normal Cell Apoptotic Cell,Cytoplasm Externalization of Cytoplasm. Phosphatidylserine, Annexin V A Key Protein in Apoptosis Signaling BD Biosciences offers Annexin V in several common formats. Changes in the plasma membrane are one of the first such as FITC PE and BD Horizon V450 for the violet laser. characteristics of the apoptotic process detected in living With the addition of these new formats more complex. cells Apoptosis can be detected by the presence of assays can be developed to look at apoptosis within. phosphatidylserine PS which is normally located on the heterogeneous cell subsets. cytoplasmic face of the plasma membrane During apoptosis. Since intracellular Annexin V is also exposed if the plasma. PS translocates to the outer leaflet of the plasma membrane. membrane is compromised a membrane impermeant dye, and can be detected by flow cytometry and cell imaging. such as 7 AAD is commonly used to distinguish between. through binding to fluorochrome labeled Annexin V when. apoptotic and dead cells to exclude the dead cells The. calcium is present, populations of cells that are stained with Annexin V only. represent the apoptotic cell populations,0 102 103 104 105. Annexin V BD Horizon V450, Radio frequency dose dependent apoptosis necrosis and cell death Low RF Field Treatment High RF Field Treatment. monitored by Annexin V BD Horizon V450 in pancreatic carcinoma. cell lines treated with a low dose of cetuximab targeted gold. nanoparticles As the RF field power increases the temperature 104 104. increases and a shift from apoptosis lower right quadrant to. frank necrosis upper left quadrant is seen, Data courtesy of ES Glazer and SA Curley MD Anderson Cancer Center. 0 102 103 104 105 0 102 103 104 105, Annexin V BD Horizon V450 Annexin V BD Horizon V450. For Research Use Only Not for use in diagnostic or therapeutic procedures 9. Tools to streamline apoptosis research,Additional Techniques for the. Detection of Apoptosis, There are many apoptosis triggers including certain Increases in mitochondrial membrane potential lead to. cytokines protein protein interactions and chemicals Once increased mitochondrial membrane permeability and the. apoptosis starts changes in the mitochondria membrane release of soluble proteins such as cytochrome c and. potential can be measured by flow cytometry using the pro caspases. BD MitoScreen JC 1 flow cytometry kit, Caspases are a series of proteases activated upon cleavage. at aspartate residues during earliest stages of apoptosis. Growth Factors,TNF FasL Cytokines,Extracellular Space. Active caspases can then cleave many proteins including. RTK Poly ADP ribose polymerase PARP and other caspases. Ras DNA fragmentation is one of the last phases in apoptosis. Cytoplasm DNA Damage, resulting from the activation of endonucleases during the. apoptotic process There are several established methods. Caspace 1 2 p90RSK, for the study of DNA fragmentation including isolation and. Mitochondria, Bcl 2 BAD Cdc2 separation of DNA fragments by agarose gel electrophoresis. and end labeling,BD Cell Pathways tool BAX, To support signaling research the BD HtrA2 Cytochrome. Diablo AIF,Biosciences website includes the BD Cell. Pathways tool powered by Ingenuity HtrA2,Diablo Cytochrome. Systems to help researchers explore the C,pathways that involve target molecules Apaf1. of interest AIF Endo G,Caspace Caspace Caspace,PARP Lamin. Acinus ROCK1 Gas2 Fodrin A, DNA Chromatin DNA Cell Shrinkage Caspace independent. Repair Condensation Fragmentation Membrane Blebbing DNA Fragmentation. Feature Measured Assays Key Features, Plasma Membrane Alterations Annexin binding assay Detects early apoptosis markers. Phosphatidylserine Exposure Single conjugates Quick and easy. Annexin V kits Flow cytometry or immunofluorescence application. Mitochondrial Changes BD MitoScreen Kit Fast easy single cell resolution by flow cytometry or fluorescent. microscopy, Caspase Activation Caspase Activity Assay Kits and Reagents Quick and easy uses spectrofluorometry. Active Caspase 3 immunoassays ELISA flow cytometry or Western blot. DNA Fragmentation APO BrdU TUNEL Assay Works with adherent cells single cell resolution in conjunction. APO DIRECT TUNEL Assay with cell cycle analysis by flow cytometry. With an overwhelming number of available techniques and products selecting the most appropriate. method is often difficult To help make this choice easier the overview above summarizes commercially. available assays from a biological perspective, 10 For Research Use Only Not for use in diagnostic or therapeutic procedures. Active Caspase 3 DEVD AMC DEVD AMC,Inhibition,Measurement of Cleaved Caspases and PARP. Active Caspase 3 DEVD CHO DEVD CHO DEVD AMC No signal. Caspases are important initiators of apoptosis One of the. earliest and most consistently observed characteristics of. apoptosis is the activation of a series of cytosolic proteases. called caspases When apoptosis is activated caspases cleave. multiple protein substrates en masse which leads to the. loss of cellular structure and function and ultimately results Caspase 3 cleavage inhibition reactions. Active caspase 3 binds to the fluorogenic Ac DEVD AMC. in cell death 4 In particular caspases 8 9 and 3 have been. substrate and cleaves it between asparatic acid D and. implicated in apoptosis caspase 9 in the mitochondrial AMC releasing the fluorescent AMC AMC fluorescence is. pathway caspase 8 in the Fas CD95 pathway and caspase 3 quantified by UV spectrofluorometry The Ac DEVD CHO. more downstream activated by multiple pathways aldehyde inhibitor binds strongly to the caspase 3 active. site and blocks substrate binding Hence Ac DEVD AMC is. BD Biosciences carries a variety of reagents to measure not cleaved and fluorescence is not emitted. caspases particularly caspase 3 They include antibodies. directed exclusively against the active form of the caspase. These antibodies are available in a variety of formats and can. be used for flow cytometry imaging ELISA and Western blot. BD Biosciences offers a range of tools for caspase activity. assays from individual fluorogenic peptide substrates and. inhibitors to kits to ready to use assay plates All are based. on the use of synthetic tetrapeptide substrates5 that are. designed such that proteolytic cleavage by active human. or mouse caspases results in release of a fluorophore. or chromophore The individual synthetic tetrapeptide. substrates together with the caspase inhibitors and active. caspase enzymes offer flexibility in the experimental Untreated Untreated. design of a caspase activity assay A B,Relative Cell Number. Flow cytometric analysis of apoptotic and non apoptotic. populations using anti active caspase 3 antibodies. Jurkat T cells A A1 or mouse thymocytes B B1 were left 101 102 103 101 102 103. untreated A B or treated for 4 h with camptothecin A1. or a mouse Fas monoclonal antibody clone Jo2 Cat No Treated with Camptothecin 4 h Treated with mouse Fas mAb clone Jo2 6 h. 554254 to induce apoptosis B1 Cells were permeabilized A1 B1. Relative Cell Number, and then stained with PE conjugated active caspase 3. antibodies Cat No 557091 Untreated cells were primarily. negative for the presence of active caspase 3 whereas. about half of each population of cells induced to undergo 55. apoptosis had detectable active caspase 3,101 102 1033. 10 101 102 103, For Research Use Only Not for use in diagnostic or therapeutic procedures 11. Obtain the complete picture,Additional Proteins for the. Study of Apoptosis, In addition to caspases and Annexin V there are several other The TNFR family contains many members including CD95. proteins important for the study of apoptosis including the that can be divided into three major groups based on. Bcl 2 family tumor necrosis factor receptor TNFR family structure Signaling through the TNFR pathway leads to. PARP and other signaling molecules Bcl 2 family members apoptosis 7. identified by the presence of conserved BCL2 homology BH3. PARPs are DNA repair enzymes that are activated by DNA. domains are versatile key regulators of apoptosis Bcl 2 for. strand breaks Cleavage of PARP by caspase 3 into 24 and. example protects cells from apoptosis by associating with. 89 kDa fragments inactivates the PARP enzyme, the mitochondrial membrane and preventing the release of. cytochrome c from the mitochondria In contrast other Bcl 2 BD Biosciences carries antibodies specific for cleavage. family members such as Bax promote apoptosis Increased products of PARP that are useful markers of apoptosis. levels of Bcl 2 have been reported in cancer 6 These antibodies are available in a variety of formats and. can be combined with other markers to gain additional. information about the cell 8 9, In this experiment Jurkat cells were treated with camptothecin. a potent inhibitor of topoisomerase I and apoptosis inducer. Phosphorylation of H2AX a protein important for maintaining. genome integrity has been shown to correlate with levels of. DNA damage 10 Using multicolor flow cytometry cell proliferation. BrdU apoptosis cleaved PARP and DNA damage histone H2AX. pS140 were evaluated in the same experiment,Phospho H2AX Alexa Fluor 647. Untreated 2 5 M Camptothecin 5 M Camptothecin 10 M Camptothecin 20 M Camptothecin. 3 24 7 30 12 12 20 29 19 30,59 14 56 7 51 6 44 7 43 7. 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105. 12 For Research Use Only Not for use in diagnostic or therapeutic procedures. K E Y R E G U L AT O R S, Simultaneous Studies of Apoptosis Cell Cycle and DNA. Apoptosis and cell proliferation assays are particularly useful. for basic cancer research and drug discovery Comparing. data across different experiments can be challenging due. to variability introduced by sample handling timing and. variability within the sample, Multicolor flow cytometry addresses these challenges and B B. is an excellent tool to study apoptosis and cell proliferation. Relevant markers can be combined with cell phenotyping. markers to look at events within subpopulations of cells. Antibodies to phosphoproteins can be used to examine. phosphorylation events,Immunofluorescence of cleaved PARP. HeLa cells grown were either left untreated A or treated with staurosporine 1 0. mM 4 h to induce apoptosis B Cells were then fixed with 3 7 formaldehyde. 15 min on ice then permeabilized in 0 25 Triton X 100 3 BSA PBS 15 min. on ice Cells were then washed twice with 3 BSA PBS and stained with 4 L mL of. FITC labeled anti PARP in 3 BSA PBS 1 h at RT Cells were washed twice with 3. BSA PBS and then visualized by immunofluorescence microscopy A and B represent. phase correlates of A and B respectively The results indicate that untreated cells. were primarily negative for cleaved PARP A whereas a significant percentage of. the staurosporine treated population is positive for cleaved PARP. Cleaved Parp Alexa Fluor 647, Untreated 2 5 M Camptothecin 5 M Camptothecin 10 M Camptothecin 20 M Camptothecin. 3 1 7 3 15 6 27 9 26 10,64 32 67 23 60 19 50 14 50 14. 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105 102 103 104 105. For Research Use Only Not for use in diagnostic or therapeutic procedures 13. BD Biosciences instruments and reagents are backed For example our website bdbiosciences com now. by a world class service and support organization with incorporates BD Cell Pathways a collection of detailed. unmatched flow cytometry experience Our integrated interconnected interactive maps of biological signaling. approach combines high content bioimaging and flow and metabolic pathways Researchers can look up specific. cytometry instrumentation with trusted certified reagents genes or molecules in the knowledge database trace the. and advanced applications The BD Biosciences tools enable pathways that involve them and find BD products related. our customers to discover more and obtain the most to them. complete picture of cell function and at the same time Technical Applications Support. experience improved workflow ease of use and optimal BD Biosciences technical applications support specialists. performance are available to provide field or phone based assistance. Researchers come to BD Biosciences not only for quality and advice Expert in a diverse array of topics BD technical. products but as a trusted lab partner Our repository of application specialists are well equipped to address. in depth up to date knowledge and experience is available customer needs in both instrument and applications. to customers through comprehensive training application support. and technical support and expert field service,References. 1 Lodish H D Baltimore A Berk S L Zipursky P 5 Thornberry NA Chapman KT Nicholson DW 8 Boulares AH Yakovlev AG Ivanova V et al Role. Matsudaira J Darnell eds Cell organization Determination of caspase specificities using a of poly ADP ribose polymerase PARP cleavage. subcellular structure and cell division 1995 peptide combinatorial library Methods Enzymol in apoptosis Caspase 3 resistant PARP mutant. Molecular Cell Biology Third Edition W H 2000 322 100 110 increases rates of apoptosis in transfected cells. Freeman and Company New York pp 141 188 J Biol Chem 1999 274 22932 22940. 6 Buggins AG Pepper CJ The role of Bcl 2 family, 2 P rez Cadah a B Drobic B Davie JR H3 proteins in chronic lymphocytic leukaemia Leuk 9 Rouleau M Patel A Hendzel MJ Kaufmann SH. phosphorylation dual role in mitosis and Res 2010 34 837 842 Epub 2010 Mar 31 Review Poirier GG PARP inhibition PARP1 and beyond. interphase Biochem Cell Biol 2009 87 695 709 Nat Rev Cancer 2010 10 293 301 Epub 2010 Mar 4. 7 Russo M Mupo A Spagnuolo C Russo GL, 3 Hedrick SM Ch en IL Alves BN Intertwined Exploring death receptor pathways as selective. pathways of programmed cell death in immunity targets in cancer therapy Biochem Pharmacol 10 Tanaka T Huang X Halicka HD et al Cytometry of. Immunol Rev 2010 236 41 53 Review 2010 80 5 674 682 Epub 2010 Mar 17 Review ATM activation and histone H2AX phosphorylation. to estimate extent of DNA damage induced by,4 Salvesen GS Riedl SJ Caspase mechanisms Adv Exp. exogenous agents Cytometry A 2007 71 648 661,Med Biol 2008 615 13 23 Review. 14 For Research Use Only Not for use in diagnostic or therapeutic procedures.