2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Introductions and Applications of Cell Function Assay Jin HAO Application Specialist, BD Biosciences Jin.Hao 1 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2 Fairlegh S. DickinsonMaxwell W. Becton BD, founded in 1897, is a global medical technology company that is advancing the world of health by improving medical discovery, diagnostics and the delivery of care. BD has more than 45,000 associates across 50 countries who work in close collaboration with customers and partners to help enhance outcomes, lower health care delivery costs, increase efficiencies, improve health care safety and expand access to health. 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 3 给药流程解决方案 MPS 呼吸解决方案 RS 糖尿病护理 DC 制药系统 PS 药物制备及分发技术 DPT 诊断系统 DS 生物科学 BDB 标本分析前处理系统 PAS 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 4 BD FACSCelesta Research platform 10C 12C 14C solution 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Citations of Flow Cytometers 5 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD Pharmingen Total Solution 6 T/B cell research CBA-Cytometric Bead Array IMag Cell Separation System Cell function assay Stem Cell ResearchBD Phosflow ELISA/ELISOPT WB/IHC/IP/IF/Bioimaging BD Pharmingen 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. BD PMG Produce Line 7 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Cell Function Assay Landscape 8 DNA Fragmentation Phosphatidyl Serine Exposure Mitochondrial Membrane Potential Caspase activity Esterase Activity Membrane Integrity DNA Content Proliferation Dye Retention Ki-67 Status DNA Content BrdUIncorporation Cyclin Expression Histone H3 Phosphorylation Live Cells Fixed Cells Live Cells Fixed Cells Live Cells Fixed Cells Apoptosis Viability Proliferation and Cell Cycle Cellular Process 556570 FITC Annexin V Apoptosis Kit 551302 MitoScreen JC-1 550914 PE Active Caspase-3 Apoptosis Kit 556405 APO-BRDU Kit 559925 7-AAD 562247 Fixable Viability Stain 450 561908 Hoechst 33342 Solution 565082 CFSE 562899 BV421 Mouse Anti-Ki-67 340242 Cycletest Plus DNA Reagent Kit 557892 APC BrdU Flow Kit 554109 FITC Mouse Anti-Human Cyclin D1 Set 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Apoptosis 9 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 细胞凋亡，又称为程序性细胞死亡 (Programmed Cell Death) 是机体 主动的、高度有序地清除无用细胞的过程。 Apoptosis 10 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Apoptosis 11 1. Phosphotidyl Serine exposed 2. Mitochondrial potential decreases 3. Caspases activated 4. DNA fragmentation 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Apoptosis: Annexin V mechanism 12 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Annexin V/PI detect apoptosis 13 Live cells: Annexin V Negative PI Negative Early Apoptotic: Annexin V Positive PI Negative Late apoptotic/dead: Annexin V Positive PI Positive BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (Cat.No. 556547) on Accuri C6 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1X106 cells/ml Transfer 100ul of the solution to a 5ml culture tube Add 5ul of FITC Annexin V and 5ul PI Gently vortex the cells and incubate for 15min at RT in the dark Add 400ul of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1hr. Protocol for Annexin V/PI 14 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Optimized for suspension cells Compatible across most species Compatible for cell surface staining as long as the calcium concentration is maintained Using Binding buffer during the whole assay Check trypsin/EDTA for adherent cells Calculate compensation using single stained apoptotic cells Tricky to determine FITC-%PE compensation value sometime Negative group and biological control group PE Annexin V and APC Annexin V for GFP transfected cell lines Tips: 15 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Apoptosis 16 1. Phosphotidyl Serine exposed 2. Mitochondrial potential decreases 3. Caspases activated 4. DNA fragmentation 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. Apoptosis: Mitochondrial membrane potential 17 BD