How to define flow cytometry? Flow cytometry. Historic. Flow cytometry principle. Flow cytometers: analysis and cell sorting Influx Becton Dickinson

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1 How to define flow cytometry? Méthodes d étude de la cellule MV426 Flow cytometry The possibility to measure individually and simultaneously several physical and biological characteristics of a cell in a heterogeneous solution. It allows the identification of subpopulations and the estimation of an average population To measure (-metry) optical cells properties (cyto-) through a fluid (flow) in front of an excitatory laser beam High rate (5.1 4 cells/s) and high sensitivity (1 antigenic factors/cell) The possibility of separating physically every cell analysed Historic 1934 Moldavan - Cellular numeration : capillary + photoelectrique captor 1949 Coulter - Count, size (conductance variability) 1953 Crosland -Taylor - Use laminar flow as describes by Reynolds in 1883 CyFlow Cube - Partec LSR Fortessa Becton Dickinson 1965 Kamentsky - Analysis of cells constituents (2 parameters : DNA + Proteins) Fulwyler - Cell sorting using electrostatic methods 1969 Van Dilla - Laser excitation Some examples of Gallios Beckman Coulter Flow cytometers : analysis 1972 Herzenberg - Cells sorting (1st article et 1st cytometers commercialization) 1978 Schlossman - Monoclonal antibodies production Reinhertz - New fluorochromes development 24 Perfetto - Analyze of 19 parameters (17 fluorescent signals) 26 Chattopadhyay - Immunophenotypage by quantum Dots MACSQuant MiltenyiBiotec EasyCyte8HT Guava Technologies C6 Accuri MoFlo XDP Beckman Coulter Flow cytometry principle Astrios Beckman Coulter Some examples of Flow cytometers: analysis and cell sorting Influx Becton Dickinson 1) Fluidic system Presents Particles in front of the laser beam 3) Electronic system Allows the conversion from the different luminous signals to electronic signals which can be stored and processed by the computer via an appropriate software FACSAria Becton Dickinson FACSJazz Becton Dickinson Invitrogen 2) Optical system Composed of one (or more) monochromatic excitation source(s) (LASER), optical filters and mirrors which select and separate emission wavelength towards the appropriate detectors

2 Sample under pressure Cells suspension sheath fluid under pressure Carry away Flow Cell Nozzel Cell flow Light excitation (laser(s), lamp) Fluidic system Carry vein hydrofocalization Separation Alignment Laminar flow Sheath fluid flow induces a fast and regular acceleration to cells and forces them to be aligned in order to be analyzed one by one Light excitation Psa low Psa Ps Weak pressure difference Slow speed of passage Important focalization of cells Tiny distribution Sample pressure Sample under pressure Cells suspension sheath fluid under pressure Flow cell Light excitation Psa high Psa Ps Important pressure difference Fast speed of passage Weak focalization of cells Large distribution Psa = pression sample Ps = pression sheath The cytometer records the behaviour of the cell trough the laser beam, by measuring : Optical system Light scattering (informs on the morphology and cell structure) Detection at 9 granularity: SSC or SS or 9LS Optical system Fluorescence emission («Fluorescence Light» (FL)) Spontaneous (autofluorescence) Associated to free or bound fluorochromes Fluorescence are emitted in all directions and are always in another color than the excitation laser Light excitation Detection at small angles size : FSC or FS or FALS Invitrogen Invitrogen Color depends of the fluorescent probe nature Invitrogen - Light scattering at small angles (Forward Scattered Light, FSC) represents the light incidence angles from the LASER and is directly proportional to the particle size and area (relative measure of size) - Light scattering at large angles (Side Scattered Light, SSC) measures the reflected light and depends on granularity and complexity of the particle (relative measure of granularity) Induced fluorescence from different fluorochromes is separated by an optical filters plot. The selection filters depends on the Excitation and emission wavelengths Optical system Optical filter Filters and dichroic mirrors are part of optic elements. They realize the separation of channels and select wavelength Optical filters are materials which absorb specific wavelength and transmit others Incidence ray Transmitted light Absorbe light FSC signals are received by a photodiode. SSC signals and fluorescents emissions are collected and diverted to photomultipliers tubes (PMT). Each PMT have an optic filter which allow to detect specific wavelength When filter and incidence light make a 45 angle the not absorbed wavelengths are reflected Dichroic mirrors Reflected light Incidence ray Transmitted light Each fluorochrome is detected in a unique fluorescence optic channel: emitted light follows a specific path from the interrogation point to the detector Reflection Transmission Absorption 45

3 Volt Volt Height (H) % of transmission % de transmission % de transmission SP5 LP5 5/5 % of transmission % de transmission % de transmission Optical filter Optical filter Different filters types used in CMF Different filters types used in CMF l >5 Short Pass filter Long pass filter Band Pass filter l >525 Short Pass filter Long pass filter Band Pass filter l < l > l <5 l <475 Transitions between absorption and transmission are not perfect SP filter LP filter BP filter example SP 52 LP 52 BP 5 BP 5/ wavelength l wavelength l wavelength l wavelength l wavelength l wavelength l Optical filter Optical system Different filter types used in CMF Band Pass 525/ BP 525/ nm Ex : Optical bench Ligth sources Light source Dichroïc Long Pass wavelength l BD LSR cell analyzers Invitrogen Light source wavelength l BC Gallios BC MoFlo XDP A. Munier-1/212 Photons detectors In order to measure the optical signals they have to be converted in electric signals (volts). The choice of the detector depends on the photon number Diodes 1v 1e- Optical system Detect intense signals (ex : size parameter) Impulse formation Flow Laser Electronic system The electrical signals created by light emission are called impulsions. Their amplitude values lies between and 1 volts 1 Area (A) Laser Photomultipliers Tubes (PMT) 1v ne- High sensitivity (ex : structural and fluorescent parameters detector) used for weak signals Laser Width (W) Time (µ Seconds) Time

4 SSC SSC Cells number Cells number Cells number Electronic system Electronic system electrical impulsion (volt) has to be transformed into numerical data by a digital analogical converter (ADC) Intensity (volts) arbitrary units Convert into binary signal by the ADC, signals are processed individually Cells Light excitation 1 Conversion 124 ex : An ADC of 1 bits (2 1 ) enables 124 values Flow Photomultiplier Amplification Each mesurement from each detector is called to as a parameter Time Linear or Logarithmic scale etc Arbitrary units Relative light intensity Data acquisition The cytometer stores all parameters of every cell : List mode (LMD) Histogram Data representation Two parameters histogram Cells analysis FSC SSC FL1 FL2 FL3 Time (µsec) 1 rst de rd th Last Advantage : everything could be recalculated - + Relative light intensity Three-dimensional plot Other two parameters representations 842 UL UR 83 LL LR Trigger parameters Electronic treatment of the signal Threshold Threshold and trigger parameters Often done on the size, it allows to reduce the number of events without interest to not saturate the system Volt 1 Measurement of amplification Threshold level Regions permit to Isolate groups of interest, better discrimination by coloration, statistic calculations Data analysis Granulocytes Monocytes 1 Time (microsec.) Volt 1 Measurement of amplification Threshold level FSC Lymphocytes 1 Time (microsec.) FSC

5 Intensity Fluorescence Peak events events Data analysis Data analysis Gates Permit to combine logical regions, to define subgroups and to restrict the analyze on interesting cells signals to characterize them (statistics) R1 R1 R2 G /G 1 G 2 /M Ungated Counts A few examples 481 R1 mean : 6,45 36 R1 R1 : 68,94% R2 mean : 333,75 R2 : 3,96% 24 FL2-Log_Height Comp Percentage 1 5 R2 R3 R2 : 1,29 R3 :, overlay Doublets S Gated R1 and R FL1-Log_Height 1 1 R5 R5 :,72 R4 R4 : 97, FL1-Log_Height Comp 2C 4C Booleans equations etc. AND OR NAND NOR Doublet elimination Signal diffraction, refraction and reflection Laminar flow Refraction (dead or alive) Laser Diffraction (size) Excitation light Refracted light G1 G2 or M Doublet G1 2 G2 Excitation light Refraction is principally between 2 and 12 angles: FSC 1 G1 2 x G1 2n Reflection (cell surface, nucleus, granules ) 4n Reflected light Integral Area (relative value) Height Peak (relative value) 1 1 Temps Area Propidium Iodide Diffraction is principally between and - 4 angles: FSC Excitation light Reflected light Reflection is principally at 9 : SSC Physical properties Discrimination between living and dead cells Granulocytes Deaths Cells Homogeneous cell size Living cells Forward scatter Monocytes Heterogeneous cell size Lymphocytes Forward scatter

6 Intensity Fluorochrome characteristics Fluorochrome Molecule induces an electronic transition which then induces fluorescence. Associated to a macromolecule, it is called fluorophore Brightness fluorochromes Extinct coefficient multiplied by quantum output Excitation spectrum are compatible with the light source ABS EM wavelegnth (l) Quantum output Ratio between the number of emitted photons and the number of absorbed photons CD8-PerCP CD8-FITC CD8-ECD CD8-APC Extinct coefficient e Measurement of a reference fluorophore at a wavelength at 1M in a 1cm tank. (the more the value the more the fluorescence is elevated) Signal stability Signal stochiometry CD8-PC7 CD8-PC5 CD8-PE Choice of fluorochromes Spectral overlapping The excitation spectrum are compatible with your system BUT for multicolor is very difficult Use brightest fluorochromes for least expressed proteins Use dimmest fluorochromes for most highly expressed proteins Choose association of fluorochromes to avoid overlaps... hence limit spectral overlaps between fluorochromes and calculate compensation two methods for compensation : Medians method (manual) Slope method (automatic with new generation system) Spectral overlapping A contamination percentage of a fluorochrome by another Medians method (Manual compensation) FL1 FL2 Tubes must contain negatives cells and monomarked cells (or beads) 53/4 58/3 Photomultipliers voltage has to be high enough, allowing to have negatives cells in first decade B A Gate must include positives and negatives cells of a same autofluorescence (ex gate on lymphocytes). Adjust the compensation so as to positives and negatives cells, have same median Photomultiplier FL2 detects FITC fluorescence (A) Photomultiplier FL1 detects PE fluorescence (B) The calculated shift must be then entered in the system This procedure must be done on each fluorophore (do not forget to reset all parameters

7 Intensity Intensity Compensation Medians method A label (not a color) is attributed to each detector. The compensation method consists in removing a percentage from a signal to another signal Analog signal Compensation Fluo1 Fluo2 PE - FL2-Log_Height 53/4 58/3 1 5 R R3 Counts Fluo1 -FITC Counts Fluo2 -PE PMT Compensation etc. ADC Wavelength Fluo1 Fluo2 FL2-Log_Height Comp PE - FL2-Log_Height 53/4 58/3 R4 R FITC - FL1-Log_Height 1 5 R2 R Counts FITC - FL1-Log_Height PE - FL2-Log_Height Fluo1 -FITC Fluo2 -PE Counts Counts digital signal PMT ADC Compensation etc. Wavelength R4 R FITC FL1-Log_Height - FL1-Log_Height Comp Compensation PE-%FITC FITC - FL1-Log_Height FL2-Log_Height PE - FL2-Log_Height Comp From C. Aït Mansour - Miltenyi Compensation Log 4 decades Log 5 decades hyperlog Compensation Make compensation settings with bright fluorophores Do not touch values of PMTs after compensation settings No compensation Compensation From C. Aït Mansour - Miltenyi Required controls Dilution of Antibodies Required controls Example 5 colors N Tube FITC PE PE-TR PE-CY5 PE-CY7 1 expérimental CD3 CCR7 CD45 RO CD27 CD8 2 non coloré Ctrl isotypique t t t t t 4 monomarquage CD3 -/t -/t -/t -/t 5 monomarquage -/t CCR7 -/t -/t -/t 6 monomarquage -/t -/t CD45 RO -/t -/t 7 monomarquage -/t -/t -/t CD27 -/t 8 monomarquage -/t -/t -/t -/t CD8 Useful controls : FMO (Fluorescence minus one) 9 Ctrl régions - CCR7 CD45 RO CD27 CD8 1 Ctrl régions CD3 - CD45 RO CD27 CD8 11 Ctrl régions CD3 CCR7 - CD27 CD8 12 Ctrl régions CD3 CCR7 CD45 RO - CD8 13 Ctrl régions CD3 CCR7 CD45 RO CD27 - Titrate antibodies Secondary Ab 1/1 Secondary Ab 1/5 Primary Ab 1/25 1/5 1/1 1/2 Control with the same isotype and the same dilution as the specific antibody Cytométry 69A : (26)

8 Spreading data Unstimulated control Cytométry 69A : (26) Antigens/ receptors Excitation Emission AMCA Pacific blue FITC Sensible to ph Phycoérythrine 495, 532, PE-Rouge Texas* Red 613, ECD PE-Cyanine Tricolor, quantum red. Visible light sensibility Allophycocyanine PE-Cyanine , 532, PE-Cyanine CY7 could be altered by the fixation APC-Cy Nucleic acids Excitation Emission DAPI, Hoechst A-T bases Hoechst A-T bases, vital Sytox blue Intercalate, viability Cell trace calcein violet AM hydrophobic YO-YO ADN Acridine Orange 5 53, 64 ADN/ ARN TO-TO ADN Chromomycine G-C bases Pyronine Y ARN Propidium Iodure 536 (36 et 488) 617 Intercalate : ADN and/or ARN, viability Alexa : 35, 45, 43, 488, 532, 568, 594, 61, 633, 647, 66, 68, 7, 75 Insensible to ph, soluble in water EMA, ethydium monoazide 51 6 Viability, covalent/ photo-achievable PE/CY5 fixed monocytes and B lymphocytes of mice SJL, AKR, NOD Rappel PE-CY5 excited at 488 nm by the emit energy transferred from PE but also by the laser 633 nm via CY5, compensations could be elevated or difficult to realize so try to replace PE-CY5 by PE- CY5.5 or PE-CY7 in a combination with APC 7 A-actinomycine D G-C bases, viability Amine reactive dyes : an effective tool to discriminate live and dead cells in polychromatic flow cytometry. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roderer M,. J Immunol Metghods. 26 Jun 3;313(1-2): Cellular functions Excitation Emission Monochlorobimane glutathion INDO-1-AM / 41 Free/ bound Ca2+ Ex : Nucleic acid dies FLUO Free non fluo/ bound Ca2+ fluo Fura Red Free fluo/ bond CA2+ non fluo DIOC4 à 6, JC-1, CMXRos Rh Mitochondrial potential NAO (nonyl acridine orange) BCECF 5 52/ 62 Mitochondrial mass membrane SNARF / 67 ph6/ 7,5 CFSE ph6/ 9 FDG Cellular proliferation Fluoresceine Diacetate FDA B-galactosidase Apoptosis Excitation Emission Phiphilux Caspases-3 Annexin-V FITC Phosphatidyl-serine dutp-fitc OH DNA ends YO-PRO ADN SYTO ADN ECFP EGFP EYFP DsRed 49, mrfp Spectrum excitation Permeant Specificity Toxicity U.V : Hoechst, DAPI 488 : IP, 7-AAD 633 : TO-PRO3 Hoechst Vybrant DyeCycle Violet stain Non : IP A-T : Hoechst C-G : 7-AAD, Chromomicyne Hoechst DRAQ5

9 Cell Flow Sorting Continuous jet sorting : principle Partec System Becton System Cell Flow sorting Separate cells in a flux according to their properties Fluid Piezo-electric valve Fluorescence-Activated Cell sorting FACS is a trademark of Becton Dickinson Cells path when opened valve (unsorted cells) Collection tube Cells path when closed valve (sort) Center collection tube : sort Off center collection tube : unsorted Cf C. Jayat Atelier INSERM 13 Sort jet-in-air Electrostatic sorting nozzle stroboscope Droplet formation 1- Adjust sample and liquid sheath pressure. 2- Induce an amplitude variation and a variable frequency on the flow Drop delay Time needed for one cell to go from the analyze point to the droplet formation Deviation plates Target cell identification To analyze cells and to define sorting windows Deflected jets Droplet charge The drop charge determines the applicable tension on a droplet. Charged droplet will be deviated from the normal path Waste bin Droplet deviation An electrostatic field deviate the droplet Cell sorting jet-in-air : principle A Sort strategy B Electric impulsion Variable position of the cell in the flow (coincidence notion) Detector nozzle Laser beam Time Time 1 Time Time 1 Intersection flux / laser Drop Delay Last attached droplet (DGA)? A sorting envelop is defined depending on the direction needed for cell sorting Deflection charged plate Waste bin Collecting tubes support Purity 1 droplet Detection of coincidence ON Purity/ Output 2 droplet Detection of coincidence ON Output/ Purity 3 droplet Detection of coincidence ON Output 3 droplet Detection of coincidence OFF

10 MPM2 Alexa 488 fluorescence Cyclin A2 FITC Cell Number Cell Number Application field Studies Fundamental and applicative research Medicine (hematology, immunology, cancerology etc), Industry: food processing, environment, cosmetology, pharmacology and toxicology. Morphological analysis size, numeration, cellular viability, Antigenic analysis (membrane and cytoplasme) phenotype of blood cells, hematology, leukemia's, antigenic quantification, allergologic tests, prenatal diagnosis, specificity and antibody titration... Functional analysis cellular signalization, metabolic activity, membrane integrity, proteins and genetic expression, ionic flux, intracellular PH... Nucleic acid analysis Cell sorting DNA and RNA content, cellular cycle, proliferation, apoptosis, cellular viability... physiological criterions (fluorochromes), fluorescents telltale genes (GFP ), phases of the cellular cycle, males or female spermatozoids... Flow cytometry and safety DNA/ cell cycle Biological and chemical risks Phase G/G1 G1 44,6% S 21,8% G2+M 33,6% Linear markers give an indication but it is just an estimation Electrical risks G1 41,6% LASER risks Phase S Phase G2 + M S 28,8% G2+M 29,6% Aerosols risks Propidium Iodure Sound risks Mathematic models are more precise without being perfect MultiCycle Essential conditions for DNA content analysis Cell cycle Identification of mitosis cells G/G1 Utilization of a valid staining method for DNA Optimal use of the cytometer (marble of calibration, DNA beginning of the fluorescence, correct voltage) Apparition of the CyclineA2 during the cycle IgG1 S G2/M Passing time : 2 events per second Coefficient of variation peak GG1 < 5% Ctrl Weak fragment proportion (<2%) Eliminate aggregates M 1 cells minimum to calculate a cycle Propidium Iodide Propidium Iodide G2 Have diploid cells as reference (ex : chicken red blood cells ) DNA content PI fluorescence J. Sobczak UMR 7622 I. Gasnereau UMR 7622

11 CD56 events Cell number DNA peak Modulation of the cell cycle Divers inhibitors effects of topo-isomerases (treatment during 16 hours) Cell cycle a tumorous cell line treated by antimitotic agent % of cells in G2: Reference: 16% R1 R1 R1 R1 drug.1: 26% 2c drug.3: 5% drug.9: 83% 1c 4c Asynchrones cells J. Sobczak UMR 7622 Camptothecine 1 mm Etoposide 25 nm ICRF mm Agnès Chassevent - 29 Paul Papin Center - Angers Cell proliferation Click-it EdU follows a protocol of Aldehyde fixation and detergent permeabilization Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) Phase S G1 + G G2 + M Fix for 15 minutes, wash Permeabilize for 3 minutes, wash Incubate in click labeling cocktail for 3 minutes, wash Optimal : incubate with cell cycle stain for 15-3 minutes Analyze Control cells : EdU cells EdU-incorporated FL3-H J. Sobczak UMR 7622 Analysis of cell division Proliferation of T lymphocytes after 4 days of stimulation D D4 D6 *MDR Resistance MultiDrogue Clinical study of MDR* N 1 N 2 N 3 CFSE (carboxyfluoresceine diacetate, succimidyl ester) with verapamil without verapamil Rhodamine 123 A. Saoudi Drénou, B

12 Propidum iodide # Cells GFP FL1-H Ki-67 FITC Ki-67 FITC # Cells Effect of transfected gene on cell cycle Ki-67 /DNA transfected gene % 32% of cells Ki FL4-H Hoechst FL4-H Hoechst % Propidium Iodide (DNA) Propidium Iodide (DNA) Derek Davies, Cancer Research UK Agnès Chassevent - 29 Paul Papin Center Angers Apoptotic event Exposure of phosphatidylserine to external face cytoplasmic membrane Apoptotic event intracellulare Ca++ increase Indo1 emits blue fluorescence when it is free Indo 1 emits purple fluorescence when it is binding Ca++ They measure ratio indo bind/indo free according to time T human lymphocytes non stimulated T human lymphocytes antigen timulated Annexin V- FITC S. Valitutti Apoptotic event TUNEL (TdT-mediated X-dUTP nick end labelling) pic sub-g1 DNA Fragmentation 2n 4n J.F. Mayol CRSSA - Grenoble

13 FL3 - Beads FL3 - Beads events events 6 Stem cells DNA/ Cell cycle - Sorting Side Population (bone marrow of mouse) Hoechst (laser UV or violet) emission : blue AND red Muscle G /G 1 Apo 33% G /G 1 47% S 14% G 2 +M 6% Hematopoietic Apo. S G 2+M Adipocytes Osteoblastes Propidium Iodide 124 G /G 1 Overlay after sorting Endotheliales S G 2 +M Detection based on efflux Hoechst M.C. Gendron Propidium Iodide Multicolor analysis Multicolor 6 coulors 2 6 population CD3 FITC CD38 PE CD45 PerCP-C5.5 CD4 PE-Cy7 CD19 APC CD8 APC-Cy7 after sorting Multiplex Detection of small events Ex : Cytometry Bead Area BD Biosciences assays on beads New cytometers ail-8-pe ail-1b-pe Different Intensity of fluorescence read on FL3 ail-6-pe sensitivity, optic, electronic, informatic Detection of small particles (.2 µm) according to their size and their fluorescences and of intensity of their fluorescence ail-1-pe atnf-a-pe ail-12-pe Fluorescences probes FL2 - PE FL2 - PE Ann Biol Clin, vol. 67, n 4, juillet-aout 29

14 Detection of microorganisms Detection of microorganisms Resolution of multiple small species by forward and side scatter Chlorophylle Phycoerythrine Phycocyanine Picoeucaryotes Autofluorescence Synechococcus Prochlorococcus Marine Biology Medical Biology Crucial role of microorganisms in the functioning of earth s biosphere No Antibiotic Antibiotic added Examples of phytoplancton population analysis coming from different coastal ecosystems with each cell being identified by their light scattering properties (size) and by their red chlorophyllfluorescence excited with a 488 nm laser. FS vs SS FS vs SS Light scatter cytograms of E.coli cells before and after addition of ampicillin. Data obtained from, Bio-Rad, USA. Food Biology Plant Biology DNA in plants Detection of Listeria in milk products with CMF using fluorescent antibodies automatical analysis methods in milk products Listeria monocytogenes Not just pathogenics... Viability studies of beer yeast used in australian brewing industry S.J. Ochatt

15 Supplementary methods A hybrid Quantification Discrimination Visualization Photobleaching Reproductibility Observed events Cell sorting Complementarity Flow cytometry +++ individual YES Microscopy +/- individual Some hundreds +/- YES The ImageStream is a high-speed automated microscope that captures images in flow (low rate) ImageStreamX Amnis cells in flow Brightfield illuminator laser detector autofocus Spectral decomposition element Velocity detector Technological innovation LEAP is an imaging cell sorter (shooting laser) Technological innovation Attune a cytometer an acoustical focusing LEAP Cyntellect Attune Life Technologies Schematic of acoustic focusing principle. Technological innovation CyTOF : metal-labeled cells are introduced individually into an Inductively Coupled Plasma Cells are atomized and ionized in an argon plasma Atomic ions are extracted into the ion optics and Time-Of-Flight region They are separated by mass and counted Quantum Dots, are semiconductors whose electronic characteristics are closely related to the size of individual crystal. Excitation par UV Perspective Analysis of up to 1 stable isotope labels in a single cell No overlap between detection channels, no need to compensate No autofluorescence An unlabeled cell is invisible to the system 1 cells/ sec Efficiency of the cell intruction system 2-3% Data output in text and FCS standards The smaller dot, the closer it is to the blue of the spectrum, and the larger dot, the closer to the red. Dots can exciting by U.V or another laser. CyTOF system DVS Sciences Bandura, D.R., et al. Anal Chem 81 (16): , 29. Quantum Dot Corporation

16 Recent technological developments Conclusion Sensitivity increase and multiparameters «ELISA on beads» for detection and quantification of several products in the same sample Panel of cytokines in plasma Receptor in supernatant or in cell lysate RNA messengers «microarrays» on beads CMF is a technology turned to the polychromy Sensitivity increase and number of analyzed parameters open prospects into studies of microparticules Current technological developments QuantumDots (nanocristaux) Documentation Books and journals La cytométrie en flux. X. Ronot, D. Grunwald, J.F. Mayol & J. Boutennat. Tec et Doc. Lavoisier. Paris. 26. Practical flow cytometry. Shapiro H.M. 4 ème édition. 23. Wiley-Liss ed. Cycle cellulaire et cytométrie en flux. Grunwald, J.F. Mayol & X. Ronot. Tec et Doc. Lavoisier. Paris. 21. Cytometry : Articles Documentation Hoffman RA,Wang L, Bigos M, NolanJP : NIST/ISAC standardization study : variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads. Cytometry. 81A : (212) Zbigniew Darzynkiewicz : Critical Aspects in Analysis of Cellular DNA Content. Curr Protoc Cytom. 211 April; CHAPTER: Unit 7.2. (211) Roderer : Optimizing a multicolor immunophenotyping assay. Clin Lab Med 27 : (27) Roederer : Quantum dot semiconductor nanocrystal for immunophenotyping by polychromatic flow cytometry. Nat Med. Aug ; 12(8) : (26) Maecker HT, Trotter J : Flow Cytometry Controls, Instrument Setup, and the Determination of Positivity.. Cytometry 69A : (26) Perfetto, S. P., Chattopadhyay, P. K. and Roederer, M. : "17-Color Flow Cytometry: Unraveling the Immune System." Nat Rev Immunol 4: (24) Robert P. Wersto, et al : Doublet Discrimination in DNA Cell-Cycle Analysis. Cytometry. 46A : (21) SPACE Documentation Web cytometry Association Française de Cytométrie : OCEAN International Society for Analytical Cytology : Life technologies : Analyse cellulaire Flow cytometry resources flow cytometry tutorials EARTH List of discussion TARA Liste de discussion : Free software WEASEL :