Flow Cytometry. flow cytometer DNA apoptosis ph
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1 Flow Cytometry flow cytometer DNA apoptosis ph flow cytometry flow cytometer (a) (b) cell counting instrument (c) 1960 ink-jet technology fluorescence microscopy fluorescent dye DNA polyclonal antibody Albert Coons photodetector Köhler Milstein 1984 monoclonal antibody specificity (homogeneity) binding 1934 Montreal Andrew Moldavan static microscopy flowing system 249
2 neutral red photodetector Andrew Moldavan 1960 Louis Kamentsky Moldavan microscope-based spectrophotometer 500 ultraviolet absorption scatter of blue light 1967 Kamentsky Melamed sorting system 1969 Münster Dittrich Göhde DNA ethidium bromide Nallace Coulter Coulter Technology 1950 Coulter liquid system orifice isotonic saline solution electrical resistance Coulter (a) (b) (c) Fulwyler Coulter technology RG Sweet ink jet technology sorting cell sorter vibration of nozzle stream drop Fulwyler Coulter coulter volume 2 Crosland-Taylor laminar flow flow system 2 hydrodynamic focusing 1969 Los Alamos Marvin Van Dilla cytometer argon laser hydrodynamic focusing Fulwyler s sorter FACS fluorescence activated cell sorter Stanford Herzenberg 250
3 multiple parameter fluorescence light scatter coulter volume cell sorting FACS FACS cytometer Coulter Becton Dickinson Coulter Technology Stanford Flow Cytometry flow cytometry fluid stream flow cytometry FACS scanner vibration nozzle cell sorter FACS sorter sorting scan scan sorting FACS 3cs fluidics optics electronics 1. fluidics 3 (a) fluid flow hydrodynamic focusing flow chamber Flow chamber laminar flow turbulent flow 251
4 laminar flow turbulent flow (b) fluidic fluidic sheath fluid reservoir sheath fluid flow chamber sample tube sheath fluid flow chamber analytic chamber vibration nozzle sheath fluid intersection point fluidic A B C flow chamber 252
5 2. optics and electronics 0.5 o 5 o forward scatter FSC 15 o 150 o side scatter right-angle scatter SSC fluorochrome FSC SSC FITC anti-cd3 PE anti-cd4 488 nm argon laser FSC SSC 520 nm FITC 576 nm PE 4 parameter sorting 253
6 (a) illumination laser (b) collection optics detection device lens filter FSC PIN diodes SSC photomultiplier tubes PMTs (c) Optical filtration arc lamp filter argon laser, λ 488 nm output light filter scattered light fluorescence dichroic mirror 450 nm long pass dichroic mirror 450 nm 450 nm 450 nm short pass 450 nm 450 nm bandpass filter 530/30 nm 515~545 nm bandpass filter detector FITC PE emission bandpass 254
7 filter Fluorescein (FITC) pycoerythrin (PE) fluorescein (FITC) pycoerythrin (PE) SSC(RALS) FSC(FALS) FALS/RALS dot 255
8 SSC(RALS) SSC(RALS) FSC(FALS) FSC(FALS) contour plot density plot 256
9 (d) analysis and display histogram y parameter events SSC FSC histogram SSC FSC parameter dot contour A density B dot plot contour plot density plot G M L FSC FALS forward angle light scatter SSC RALS right angle light scatter (e) cell sorting FACS sorter parameter parameter tube cyclone 96 microtitration plate cell sorter well limiting dilution DNA transfection stable clone Cell sorter ink-jet graphic printing electrostatic deflection nozzle stream orifice break-off flow cytometer > 99% sorting fluorescence fluorochromes excitation status ground status excitation maximum emission maximum fluorochromes excitation maximum emission maximum 257
10 nm nm nm Alexa Fluor Green A Allophycocyanine H Allophycocyanin-cyanine7 conjugate H Biodipy-FL A Fluorescein A Peridinin-chlorophyll protein A Phycoerythrin-cyanine5 conjugate A Phycoerythrin-cyanine7 conjugate A Phycoerythrin-Texas Red conjugate A R-phycoerythrin 564, a A Argon-ion, H helium-neon. fluorochromes 1. fluorochromes fluorochromes laser ploidity cell cycle DNA nm nm nm 7-Aminoactinomycin D or 488A Acridine Orange (DNA) 488A 640(RNA) Chromomycin A A DAPI UV A or HeCd Ethidium bromide 493, A Hoechst UV A or HeCd LDS A Propidium iodide 495, A Pyronin Y or 488A Thiazole Orange A YO-PRO A TO-PRO H a A Argon-ion, HeCd helium-cadmium 258
11 2. nm nm nm DiOC 6 (3) A DiOC 5 (3) A di-ba-c 4 (3) or 514A DiSBaC 2 (3) A Merocyanine A MitoTracker Red CMXRos or 514A JC-1 -monomer A -dimer ~ A Rhodamine A 3. flow cytometry Indo-1 UV Indo-1 emission spectrophotometer Fura-2 flow cytometry nm nm nm CM-Dil or 514A CMTMR or 514A DiOC18(3) A Fluo A Fura Red 436/ /657 b 488A lndo /475 a UV A or HeCd MitoTracker Green FM A Nile Red A PKH or 514A Rhodamine A SNARF /630 b 488 or 514A a Dependent on Ca 2 concentration b Dependent on ph 4. Nile Red lipid oil droplet PKH26 CM-Dil DiOC18 259
12 plasma membrane PKH26 PKH67 T CTL assay chromium release assay 5. CMFDA CSFE PKH 6. SNARF seminaphthorhodafluors Molecular Probe Inc. ph SNARF 488 nm emission 580 nm ph6 640 nm ph9 emission ph ph Dihydrorhodamine 123 dihydro-ethidium rhodamine 123 ethidium DNA Flow Cytometer 1975 Köhler Milstein flow cytometer cell sorter flow cytometer fixation sorting beta-galactosidase lac Z green fluorescent protein GFP transfect lac Z GFP flow cytometer DNA DNA flow cytometer flow cytometer flow cytometer (1-4) flow cytometer shareware fluorochromes flow cytometer 260
13 1. Givan, A. L. (2001). Flow Cytometry: First Principles 2 nd Ed. Wiley-Liss. 2. Shapiro, H.M. (1995). Practical Flow Cytometry, 3 rd Ed. Wiley-Liss. 3. Darzynkiewicz et al. (1994). Flow Cytometry, 2 nd Ed. Academic Press. 4. Ormerod, M. G. (2000). Flow Cytometry, 3 rd Ed. Oxford University Press. FACS Becktmen Coulter Flow Cytomers Beckton Dickionson Flow Cytometers 4. FACS Softwares: Cytomation 5. FACS Softwares: Tree Star Inc Free Flow Cytometry Softwares: 7. MoFlow Cell Sorters 8. Molecular Probes Inc Table of Fluorochrome 261
14 262
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