UNIVERSITY OF PÉCS MEDICAL SCHOOL FLOW CYTOMETRY AND CELL SEPARATION BIOPHYSICS 2. 2015 4th March Dr. Beáta Bugyi Department of Biophysics Flow cytometry and cell separation FLOW = STREAM OF FLUID in a flowing heterogeneous particle suspension CYTO = CELL separate particles (e.g. cells) METRY = MEASUREMENT and their individual characteristics can be measured based on optical (light) signals (number, size, complexity, presence and amount of special markers) SEPARATION = SORTING on the basis of the measured parameters subsets of interesting particles can be separated from each other for further purposes (morphological, biochemical analysis, cell culturing) Workflow Flow cytometer investigated object: suspension of particles in size of 0.2 150 µm (biological particles: cells, microorganisms, blood-, bone-marrow sample ) instrument: flow cytometer measured quantities: optical signal: light scattering, fluorescence emission information/result: physical, chemical, biological features (size, complexity, presence of special markers) 1

Flow cytometer Flow cytometer 1. FLUIDICS flow and hydrodynamic focusing of particles LIGHT SOURCE lasers 4. SORTING separation of cells 3. ELECTRONICS, DATA ANALYSIS conversion of lightsignal to digital data, analysis FLUORESCENCE EMISSION (FL) SIDE SCATTER (SSC) 2. OPTICS light source, filters, mirrors, detectors FORWARD SCATTER (FSC) 1. Flow system, fluidics: flow and focusing of particles 2. Optics: light source, optical elements (filters, mirrors), detectors 3. Electronics and data analysis: conversion of light to digital signal (numbers), analysis 4. Cell sorting: separation of cells Flow system hydrodynamic focusing Hydrodynamic focusing sample fluid (particlesuspension, 10 5-10 6 cell/ml, v= 10 m/s) LAMINAR FLOW! (the two fluids cannot mix) FLOW HEAD nozzleaperture d = 50-200 µm sheathfluid (surrounds the sample fluid to protect cells from damage, distilled water, isotonic solution) interrogation point particlesare separated and alignedin a single layer of fluid particles are passed through the light beamone ata time(10 4-10 5 particle/s) Both the size and the geometry of the flow head (sample-, sheath fluid, nozzle aperture), and the characteristics of the flow (pressure) are set to establish a stable laminar flow (the two fluid do not mix), which alignes the particles and they leave the flow head one after another. 2

Flow head Optics: measurable quantities ligh scattering, fluorescence emission FORWARD SCATTER FSC: forward angle light scatter smaller angles size index of refraction LIGHT SCATTERING SIDE SCATTER SSC: side light scatter larger angles intracellular structural complexity granularity FLUORESCENCE EMISSION FL1, FL2, special parameter fluorophore specifically bound to cells, cellular components Optics: detection forward angle-, side light scatter Optics: detection forward angle light scatter DETECTOR photodiode, photoelectron multiplyer cell detector forward scatter SSC FORWARD ANGLE SCATTER detector parallel to the light source laser plate obscuration bar FSC SIDE SCATTER detector perpendicular to the light source 90 o LIGHT SOURCE (laser) Animáció: http://media.invitrogen.com.edgesuite.net/tutorials/4intro_flow/player.html 3

Optics: detection fluorescence emission FL4 FL3 FL2 FL1 DICHROIC MIRRORS: wavelength dependent absorption and reflection OPTICAL FILTERS: wavelenght dependent absorption Separation of different fluorescence emission: particular wavelenght ranges are deliverd to the appropriate detector. list mode LIST MODE: presentation of all the measured data corresponding to every single particle (raw data stored in the computer) cell measured parameter FLUORESCENCE EMISSION detector perpendicular to the light source LIGHT SOURCE (laser) 90 o single parametric representation histogram HISTOGRAM: plot of the number of particles characterized by a given parameter number of particles more single parametric representation histogram FSC SSC FLUORESCENCE smaller larger növekvő méret less complex more complex fluorescent nonfluorescent less size intracellular complexity amount of fluorofor measured parameter (FSC, SSC, FL) 4

multiparametric representation dot plot DOT PLOT: correlation method corresponding values of two measured parameters (x, y axis) are displayed as a dot (each dot corresponds to a single particle). multiparametric representation dot plot number of cells side scatter: complexity size forward scatter: size intracellular complexity number of cells multiparametric representation dot plot multiparametric representation dot plot fluorescence FL1 FL1 + FL1 - number of cells number of cells +/- +/+ -/- -/+ FL2 - FL2 + fluorescence FL2 Example: dotplotof whitebloodcell sample (sample preparation: Sedimentation, electrophoresis) side scatter: complexity forward scatter: size LEUKOCYTE GRANULOCYTE neutrofil (50-70 %), eosinophil(2-5 %), basophil(< 1%) ~ 75 % size~ 10-15 µm highcomplexity MONOCYTE ~ 3-6 % size~ 12-15 µm mediumcomplexity LYMPHOCYTE ~ 20-40 % size~ 7-8/12-15 µm low complexity 5

others Gating GATING: definition of subpopulation of cells/particles in one-, or multiparametric representation for detailed investigation/analysis. DENSITY PLOT color ~ number of cells CONTOUR PLOT dots with the same values are bound with lines 3D PLOT z axis~ number of cells Gating Cell separation Example: ratio of helper/cytotoxict lymphocytes B cell: CD19+ T cell: CD3+ CD3+/CD4+ helper CD3+/CD8+ cytotoxic natural killer cell: CD3-/CD16+ side scatter: complexity CELL SEPARATION: separation of cell populations characterized by a given parameter (FSC, SSC, FL) for further investigation (morphological, biochemical analysis, cell culturing). forward scatter: size 6

Cell sorting Principles Applications 1 2 3 4 5 flow head delay time ++ + - -- 1. Data collection 2. Decision: based on gating, the cell population to be sorted is defined. 3. Dropformation: theflow headis shaken by a piezoelectric vibrator(30-40 khz) to decompose the fluid that leaves the head intodroplets(1 droplet= 1cell). 4. Electrostatic separation: the droplet to be separatedis chargedattheplaceof dropletformationand deflectedbyan electric field. 5. Separation: 3000 5000 cell/s Broad fields of application in medical biology (basic and applied research) clinical practice (diagnostic, therapeutic monitoring,prognosticapproaches) quantitative, qualitative analysis of cell population calculationof absolute cell number and cell ratio identification and quantificationof markers functional description of cells: cell signalling cell-cell interactions morphological studies DNA damage and repair cell cycle autophagy cell death stem cell differentiaion parasitology microbiology oncology hematology clinical immunology(targeted immunotherapy, immunedeficiency, autoimmune diseases, infections) Analysisof a blood sample from an HIV infected patient T-helper lymphocytes aretargetted bythe virus, which results in a decline in the number of T-helper cell Markers: CD4+ helpert cell CD8+ cytotoxict cell Analysisof a bloodsamplefromaleukemiapatent suffering from a B cell lineage leukemia or lymphoma B lymphocytes grow out of control and accumulatein the bone marrow and blood, where they crowd out healthy blood cells abnormal FSC-SSC dot plot decrease in T lymphocyte(cd3+) Increase in B lymphocyte(cd20+) increase in human leukocyte antigen-dr+ cells 7

CD (cluster of differentiation) protokol using antigens specifically recognising cell surface marker molecules (CD) different cell populations can be identified CD molecules can have diverse role (receptor, ligand, signalling pathways, antigen recognition, growth factor, cell adhesion) + / - symbol: marker is expressed/not expressed human cells > 360 CD marker Example: lymphoidcell differentiation CD markers lymphocytes(t cells, B cells), NK cells and dendritic cells Example: myeloid cell differentiation CD cell markers monocytes, granulocytes, platelets, erythrocytes, macrophages, dendritic cells Selectionof lymphocytesfrom leukocytes FSC/SSC dot plot, gating gating LYMPHOCYTES markers: T cellscd3+ CD3+/CD8+ cytotoxic T cells CD3+/CD4+ helper T cells SSC leukocyte FSC lymphocyte 8

Selectionof CD3+ T-lymphocytesfrom lymphocytes CD3 fluorescence emission histogram, gating Selectionof CD4+ helper T-lymphocytesfrom T-lymphocytes CD4-CD8 fluorescence dot plot nonfluorescent B & NK cells fluorescent T cells lymphocyte T-lymphocyte CD3 fluorescence emsission Analysisof a blood sample from an HIV infected patient Flow cytometry and cell sortin - keywords Hydrodynamic focusing Origin and importance of pptical signals (light scattering, fluorescence emission) : list mode, histogram, correlation methods: dot plot Gating Cell sorting Keywords from previous lectures: laminar flow, photomultiplier, laser, light scattering, fluorescence Keywords to coming lectures: Fluorescence microscopy: optical filters, dichroic mirrors Ultrasound: piezoelectric effect Sedimentation: preparation of blood samples 9

Suggested web resources Supplemental material https://www.youtube.com/watch?v=2p7ysj0zkio http://media.invitrogen.com.edgesuite.net/tutorials/4intro_flow/play er.html http://media.invitrogen.com.edgesuite.net/tutorials/5data_analysis/ player.html Optical filters, dichroic mirrors Photelectron multiplyer photon reaching the photocathode photoelectron(photoelectric effect) amplificationby a dynode chain anode electric current 10

Conversion of optical signal to electronic signal 11