Flow Cytometry flow cytometer DNA apoptosis ph flow cytometry flow cytometer (a) (b) cell counting instrument (c) 1960 ink-jet technology 17 19 1940 1950 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
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
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
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
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
(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
filter Fluorescein (FITC) pycoerythrin (PE) fluorescein (FITC) pycoerythrin (PE) SSC(RALS) FSC(FALS) FALS/RALS dot 255
SSC(RALS) SSC(RALS) FSC(FALS) FSC(FALS) contour plot density plot 256
(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
nm nm nm Alexa Fluor Green 491 515 488A Allophycocyanine 650 660 630H Allophycocyanin-cyanine7 conjugate 650 755 630H Biodipy-FL 503 512 488A Fluorescein 495 520 488A Peridinin-chlorophyll protein 490 677 488A Phycoerythrin-cyanine5 conjugate 495 670 488A Phycoerythrin-cyanine7 conjugate 495 755 488A Phycoerythrin-Texas Red conjugate 495 620 488A R-phycoerythrin 564,495 576 488 a A Argon-ion, H helium-neon. fluorochromes 1. fluorochromes fluorochromes laser ploidity cell cycle DNA nm nm nm 7-Aminoactinomycin D 546 647 514 or 488A Acridine Orange 503 530(DNA) 488A 640(RNA) Chromomycin A 3 430 580 457A DAPI 372 456 UV A or HeCd Ethidium bromide 493,320 637 488A Hoechst 33342 395 450 UV A or HeCd LDS 751 543 712 488A Propidium iodide 495,342 639 488A Pyronin Y 545 565 514 or 488A Thiazole Orange 509 533 488A YO-PRO-1 491 509 488A TO-PRO-3 642 661 630H a A Argon-ion, HeCd helium-cadmium 258
2. nm nm nm DiOC 6 (3) 484 501 488A DiOC 5 (3) 484 501 488A di-ba-c 4 (3) 543 590 488 or 514A DiSBaC 2 (3) 535 560 514A Merocyanine 540 500 572 488A MitoTracker Red CMXRos 578 599 488 or 514A JC-1 -monomer 510 527 488A -dimer ~585 590 488A Rhodamine 123 485 546 488A 3. flow cytometry Indo-1 UV Indo-1 emission spectrophotometer Fura-2 flow cytometry nm nm nm CM-Dil 553 570 488 or 514A CMTMR 541 565 488 or 514A DiOC18(3) 484 501 488A Fluo-3 503 526 488A Fura Red 436/472 637/657 b 488A lndo-1 349 400/475 a UV A or HeCd MitoTracker Green FM 490 516 488A Nile Red 485 525 488A PKH26 551 567 488 or 514A Rhodamine110 499 521 488A SNARF1 514 580/630 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
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
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 1. http://www.bdtwn.com.tw/bdb/index.html 2. Becktmen Coulter Flow Cytomers. http://www.beckman.com/products/instrument/flowcytometry/adc.asp 3. Beckton Dickionson Flow Cytometers http://www.bdbiosciences.com/product_spotlights/instruments_and_data_management/ 4. FACS Softwares: Cytomation http://www.treestar.com/ 5. FACS Softwares: Tree Star Inc. http://www.treestar.com/flowjo 6. Free Flow Cytometry Softwares: http://flowcyt.cyto.purdue.edu/flowcyt/software.htm 7. MoFlow Cell Sorters http://www.cytomation.com 8. Molecular Probes Inc. http://www.probes.com/ 9. Table of Fluorochrome http://pingu.salk.edu/flow/fluo.html 261
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