BD Pharmingen™ Apoptosis, DNA Damage and Cell Proliferation Kit

Kit Contents Description of Components          Material No.          Size          Storage          Vials Kit Part A                                       PerCP-Cy5.5 Mouse Anti-BrdU          51-9007682          50 test             4ºC            1   Alexa Fluor™ 647 Mouse Anti-H2AX (pS139)          51-9007683          50 test             4ºC            1   PE Mouse Anti-Cleaved PARP (Asp214) Antibody          51-9007684          50 test             4°C            1   BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution          51-2090KE          25 ml             4°C            1   BD Perm/Wash™ Buffer (10X)          51-2091KE          25 ml             4°C            2   BD Cytofix/Cytoperm™ Plus Permeabilization Buffer          51-2356KC          10 ml             4°C            1   DAPI          51-9007681          100 μl             4°C            1 Kit Part B                                         BrdU (10 mg/ml)          51-2420KC          5 mg          -80°C            5   DNase          51-2358KC          300 μl          -80°C            5 Apoptosis, DNA Damage and Cell Proliferation Kit Multiparameter flow cytometry provides a powerful tool for resolving mechanisms by which individual cells in homogenous or mixed cell populations maintain viability, enter and progress through cell cycle or undergo cell death. For this purpose, the Apoptosis, DNA Damage, and Cell Proliferation Kit was designed with the inclusion of fluorescent antibodies specific for incorporated BrdU, phosphorylated H2AX (γH2AX) and cleaved PARP. These probes along with optimized protocols enable multicolor flow cytometric analysis of proliferation, DNA damage and apoptosis, respectively, by individual cells within samples. Immunofluorescent staining of cells that have incorporated Bromodeoxyuridine (BrdU, an analog of the DNA precursor thymidine) and flow cytometric analysis provides a high resolution technique to determine the frequency and nature of individual cells that have synthesized DNA. Exposure of cells to BrdU in either in vitro or in vivo experimental model systems allows for BrdU incorporation by actively cycling cell fractions. Pulse labeling of cells with BrdU at various time points, permits the determination of cell-cycle kinetics. Flow cytometric analysis of BrdU incorporation can readily be combined with the simultaneous analysis of cellular phosphorylated H2AX and cleaved PARP levels. Phosphorylated H2AX functions to recruit and localize DNA repair proteins or cell cycle checkpoint factors to DNA-damaged sites. In this way, phosphorylated H2AX promotes DNA repair and maintains genomic stability. Double-stranded DNA breaks caused by replication errors, apoptosis, or other physiological processes (including, immunoglobulin and TCR gene recombinations) and DNA damage caused by ionizing radiation, UV light, or cytotoxic agents lead to H2AX phosphorylation on serine 139, H2AX (pS139), to induce its function. PARP (Poly [ADP-Ribose] Polymerase) is a nuclear chromatin-associated enzyme that is involved in DNA repair. During apoptosis, Caspase-3 cleaves PARP resulting in its inactivation and the inability of cells to repair DNA damage. For this reason, the 89 kDa-cleaved fragment of PARP serves as a marker of cellular apoptosis.

(RUO)

51-9007685AK/(2 to 8C)+51-9007685BK/(max.-70C)

Flow cytometry, Intracellular staining (flow cytometry) (Tested During Development)

BrdU, H2AX (pS139), Cleaved PARP (Asp214)