FLOW CYTOMETRY PRINCIPLES AND APPLICATIONS：流式细胞仪的原理与应用.ppt

PBS 7413FLOW CYTOMETRY LABORATORY COURSE OBJECTIVES UNDERSTAND CLINICAL AND RESEARCH APPLICATIONS OF FLOW CYTOMETRY TECHNOLOGY PERFORM STAINING TECHNIQUES ANALYZE DATA INTERPRET DATA AS PRESENTED IN BASIC SCIENCE AND CLINICAL SCIENCE LITERATURE Flow Cytometry Principles And Applications/facs FLOW CYTOMETRY Fluorescence activated cell sorting (FACS) is a technology that allows simultaneous measurement of multiple physical and chemical characteristics of a single cell. These measurements are made on a per cell basis at routine rates of 500 to 70,000 cells per second in a moving stream. FACS COMPONENTS LASER OPTICS FLUIDICS ELECTRONICS LASER ARGON EMISSION LINE OF 488 nm VISIBLE LIGHT OPTICS BEAM SPLITTERS DICHROIC MIRRORS LONG / SHORT PASS FILTERS BAND PASS FILTERS FLUIDICS SAMPLE FLUID SHEATH FLUID FLOW CELL NOZZLES, 70, 100, 400 um ELECTRONICS PHOTOMULTIPLIER TUBES (PMTs) PHOTODIODE MACINTOSH G3 COMPUTER DATA PARAMETERS FORWARD LIGHT SCATTER 90o OR SIDE SCATTER FLUORESCENCE EMISSION FORWARD SCATTER LIGHT SCATTER USED IS LOW ANGLE SENSITIVE TO CELL SIZE AND SURFACE AREA LIVE / DEAD DISCRIMINATION SIDE SCATTER LIGHT SCATTER USED IS 90o SENSITIVE TO INTERNAL STRUCTURES BEST ANALYSIS USING FORWARD AND SIDE SCATTER TOGETHER; ANALYSIS OF HETEROGENEOUS POPULATIONS FLUORESCENCE DETECT BINDING OF LABELED LIGAND LIVE/DEAD DETERMINATION DNA/RNA CONTENT APPLICATIONS IMMUNOPHENOTYPING CELL CYCLE ANALYSIS APOPTOSIS CELL FUNCTION CELL SORTING CELL SURFACE STAINING SINGLE CELL SUSPENSION MONOCLONAL ANTIBODY FLUOROCHROME CONJUGATED MONOCLONAL FLUORESCENCE ANTIBODY CONJUGATED SECOND REAGENT FACS ANALYSIS / CELL SORTING ADVANTAGES OF FACS PRECISION SENSITIVITY NO PHOTOBLEACHING VOLUME SINGLE CELL ANALYSIS SAMPLING STATISTICS SORT CELLS IN SEPARATE TUBES CELL C