Principles of Flow Cytometry

Klepnutm lze upravit styl pedlohy nadpis.,Klepnutm lze upravit styly pedlohy textu.,Druh rove,Tet rove,tvrt rove,Pt rove,*,Orifice = 50 to 400 ,Principles of Flow Cytometry,Quartz,nozzle,Fluorescence signals,Focalized laser beam,Injection of cells,Fluorescence,Photodiode,Light can be measured at 90 :,Side scatter + Fluorescence,Side scatter reflects the cell content,Laser,Fluorescence intensity,FITC,FITC,10,1,10,4,10,3,10,2,Relative fluorescence intensity,Number of Events,FITC,FITC,FITC,FITC,FITC,FITC,FITC,FITC,Basics of Flow Cytometry,Cells in suspension,flow in single-file through,an illuminated volume where they,scatter light and emit fluorescence,that is collected, filtered and,converted to digital values,that are stored on a computer,Fluidics,Optics,Electronics,The automated Microscope,Waste,Detector,& Counter,Sample,This primitive diagram shows the principle: Cells are passing the microscope objective, and an electronic circuit decides whether the cells is fluorescent or not. This is how a flow cytometer works!,1,Hydrodynamic focussing in the cuvette,Sheath,Sample,Sheath,Sample,Sample pressure low, small core stream. Good for DNA analysis,High sample pressure, broader core stream.,Bad for DNA analysis,LOW,HIGH,Pressure,(=,Sheath Pressure,),drives the sheath buffer through the cuvette, and,the higher pressure,in,the sample,tube(= Sample Differential),delivers the sample,to,the cuvette,.,In,the cuvette the principle,of,hydrodynamic focussing arranges the cells like pearls,on a,string before they arrive,at,the laser interception,point,for analysis,Hydrodynamic focussing cannot,separate,cell aggregates,!,Flow cytrometry is,a,technique that requires single cell suspensions,Summary,Basic opticsc,A system of prisms and lenses directs the laser light to the interrogation point in the cuvette,Laser delay,Sheath,Sample,Umouje,cross beam,kompenzaci,Vyaduje stabiln,fluidics,Summary,Excitation light is steered with prisms and lenses to the interception point,Emitted light is collected using lenses and is split up with dichroic mirrors and filters,Tasks for the electronical system,Convert the optical signals into electonic signals,(,voltage pulses,),Digitise the data,Analyse,Height,(H),Width,(W),and,Area,(A) of,the,pulse,Send,the data,to,the analysis computer,How a voltage pulse from the PMT is generated,Voltage,Laser,Laser,Laser,t,t,t,Voltage,Voltage,1.,2.,3.,Height, Area, and Width,Time,(s),Vol,tage,Pulse area,(A),Puls,e Height,(H),Pulse Width,(W),40,0,Threshold,The threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower intensity are not displayed and not recorded for later analysis.,Summary,During passing the laser voltage pulses are generated,at,the,PMT,Amplifiers enhance the signals,Only signals passing the desired threshold,(s),are analysed,and,recorded,The data are finally passed,to,the analysis computer connected,to,the cytometer,An overview,Year,Instrument,Introduced,Most Frequently,Heard Comments,1976,FACS II,When can I get one?,1991,FACS Vantage,Do you really need 5 colors?,1998,FACS Vantage SE,Do you really need 6 colors?,2000,FACS DiVa,Do you really need 8 colors?,1980,FACS IV/440,Do you really need 4 colors?,2003,FACS Aria,When can I get one?,Why always more colours?,More,informations,from Cell,Phenotyping,(Cell Surface Antigens),around,300,CD Cell Surface Antigens,Many functional populations require 5 or more surface markers to be fully distinguished,Functional Assays,Cell Cycle (PI,BrdU, Intracellular,Cyclins,),Apoptosis (,Annexin,-V, Active,Caspase,-3),Ca,+,Flux Indo-1,FuraRed,Fluoro,-4,Cytokine Production,Intracellular Signaling (,Rb phosphorylation,),Gene Reporter Molecular Assays,GFP, BFP, YFP, CFP Expression,LacZ,Expression,What are the advantages / disadvantages?,Advantages,Save Time and Samples,(1) 6-color stain = (15) 2-color stains,Exponential increase in information,Data from (1) 6-color stain (15) 2-color stains,Identify new/rare populations (0.05%),Internal controls,Problems,Must carefully choose combinations of,fluorochrome,conjugates,Not all reagents are available in all colors,Greater potential for errors in compensation,Proper controls required,Excitation- and Emission spectra of dyes for the blue laser,Stejn excitace rzn emise,Pekryv spekter,(,overlap,),Excitace jinm laserem?,Compensation,bdbiosciences /spectra /,How much compensation is correct?,PE,PE,Importance of ACCURATE Compensation,RPCI,LFC,n = negatives,d = dim positives,b = bright positives,PE-CY5-CD8,APC CD45,n,d,b,n,d,b,Uncompensated,Compensated,Over Compensated,b,n,Where is the,CD8 dull population?!,Which marker for compensation?,Small errors in compensation of a dim control (A),can result in large compensation errors with bright reagents (B & C). Use bright markers to setup proper compensation.,Hardware Compensation,How to set compensation on the instrument,Setting compensation,Prepare single stained controls that have both a positive and negative population.,Adjust the PMT voltages so that the negative population is off the axis in every channel.,Align the centers of the positive and negative cell populations by matching the median fluorescence.,-Run unstained cells,-Adjust the PMT voltages so that the negative,population is off the axis in every channel.,Setting compensation- PMT Voltage,FL1-no stain,FL2-no stain,Uncompensated,Compensated,Median values,both = 3.2,Setting compensation - FITC single stain,FL1-FITC CD3,FL2-no stain,FL2-no stain,-Run single stained control (FITC stained only),-Adjust the compensation value so that positive and negative population have the same FL2 median fluorescence intensity.,FL1-FITC CD3,Setting compensation - PE single stain,-Run single stained control (PE stained only),-Adjust the compensation value so that positive and negative population have the same FL1 median fluorescence intensity.,Compensated,Median values,both = 2.5,FL2-PE CD4,FL1-no stain,Compensation Controls,Single Stain Controls,Does not matter as long as:,The autofluorescence is the same in the negative and positive populations you are lining up.,eg, Pre-gate on lymphocytes if you are using CD8 FITC as a single stain control,The compensation values will be valid for ALL cell types, regardless of which type of cell is used to calculate the values.,The compensation is specific for the fluorochrome,not,the cell type,Single Stain Controls - Which cells?,Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sample,OR,A different antibody may be substituted, as long as it is conjugated to the same fluorochrome.,However,Single Stain Controls - which reagents?,Caveats for substituting reagents:,Controls should be as bright as possible,As bright or brighter than the experimental stains,GFP, CFSE, and FITC are NOT the same fluorochrome,even though they are all green!,With tandem dyes (Cy5PE/Cy7PE etc.) it is necessary to use the exact same reagent,spillover varies from reagent to reagent,Single Stain Controls - which reagents?,Compensation of tandem-conjugates can differ from lot to lot,Use same reagent as experimental sample,Lots positive,Small CV, bright,Some reagents wont work (IgL, EMA/PI),can mix with regular comps,Using Antibody Capture Beads,as single stained controls,Software Compensation,Automated Tools for Setting Compensation,Compensation Tools,Must have single stained controls,Software calculated compensation for you!,Easy, accurate and quick.,Makes MULTI- Color compensation possible,Software Compensation Tools,Available on new generation machines,DakoCytomations,Summit (version 4),Coulter FC500,BD Diva,Others,Post-acquisition software,FCS Express,FCS Press,WinList,FlowJo,Others,Compensation Matrix,FL1,FL2,FL3,FL1 Comp,3.96,0,FL2 Comp,27.35,5.15,FL3 Comp,0,11.18,Compensation - Automatic Method,Automatic compensation in the Diva software offers a fast, easy and reliable method to set the correct compensation.,First,select,Create Compensation Tubes,“,from the,Instrument Menu:,Compensation - Automatic Method,The software automatically creates a list of single color tubes, based on your instrument setting.,Naturally, in certain experiments you may not want to use every channel for a multicolour experiment.,You can delete any tube from this list, but the corresponding channel is later, after automatic compensation, also deleted from your instrument setting!,Take Away Lessons,Proper CONTROLS are essential,DONT compensate by eye,Use Median to adjust the populations if you must do it manually,TRUST the software to do it for you,It does it quicker and more accurately,Polychromatick cytometrie,Design experimentu a analza,Childhood Leukemia Investigation Prague -,stav imunologie,Klinika dtsk hematologie a onkologie,UK 2.LF a FN Motol,Praha,Which fluorochrome to use?,Major Factors,Fluorochrome,brightness,PerCP, APC-Cy7 FITC ,PerCP,-Cy5.5 PE-Cy7 APC = PE-Cy5 PE,Antigen density,Background staining of,mAb,Inherent background (stickiness) of,mAb,Antibody strength (Avidity),Less antibody needed = less background,Amount of compensation required between conjugates,Single or multiple laser,Comparison of the dye intensity for the same marker,Baumgarth, Roederer, JIM, 2000, A practical approach to multicolor flow cytometry for immunophenotyping,Spektra fluorochrom,bdbiosciences /spectra /,Which fluorochrome for which marker?,In general, try to use brighter,fluorochrome,conjugates for duller antibodies or lower density antigens (e.g. activation antigens such as CD80, CD86, CD25, or CD28),Use brighter reagents for staining cell populations with high,autofluorescent,backgrounds (e.g.,granulocytes,monocytes, or activated lymphocytes),Use duller conjugates (FITC or,PerCP,) for antigens expressed at high levels (e.g. B220 or CD4),Zkreslen vlivem kompenzac,PE,FITC,PE,PE-TxRed,Pesvit (spilover, spectral overlap),z PE do FITC je mal = mal kompenzace,z PE do PE-TxRed je velk = velk komp.,PE-TxRed PE = 65%,Grafick een,Loglinear transformation“,Biexponencial display“,Zkreslen vlivem kompenzac,Je teba promyslet odkud se dvat,Obvykl problmy: PE vs PE-TxRed, PE-Cy5 vs APC,Nelze pout vdy histogram,Nelze vdy pout tverce i kvadranty,Je teba promyslet jak postavit gate (kontroly FMO),V siln komp. kanlech je men rozlien a hor kvantifikace,Design experimentu,Na interpretaci dat myslet PEDEM,Bez sprvnch kontrol nkdy interpretovat nelze,Jak naloit se zkreslenm komp. dat?,Sestavit design experimentu tak,aby se pedelo potm pi analze,PE, PE-TxRed,u,CD4,PE,pos,. bunk,CD8,PE-,TxRed,nen,Jak naloit se zkreslenm?,donor“,akceptor“,Na donor pozitivnch bukch se akceptor pozitivn znak nevyskytuje/nehodnot,CD4 PE,CD8 PE-TxRed,PE, PE-TxRed,znak s nzkou,int,. do PE,Jak naloit se zkreslenm?,donor“,akceptor“,Ni intenzita donoru = ni rozptyl akceptoru,CD3 PE,PE-TxRed,PE, PE-TxRed,vysoce exprimovan znak do PE-TxRed,Jak naloit se zkreslenm?,donor“,akceptor“,CD19 PE,CD10 PE-TxRed,PE, PE-TxRed,kvalitativn znak do PE-,TxRed,Jak naloit se zkreslenm?,donor“,akceptor“,CD3 PE,CD8 PE-TxRed,780/60,735 LP,A,575/26,556 LP,D,488/10,-,F,H,695/40,655 LP,B,610/20,595 LP,C,530/30,502 LP,E,G,PE-Cy7, PE-Alexa 750,PerCP,PerCP, PC5, Tricolor,Cy-Chrome,PE-Dyomics647,PE-Texas Red,ECD,PE Dyomics590,PE,FITC,Alexa 488,SSC,488nm Blue laser octagon,720/40,680 LP,A,660/20,-,B,C,Alexa 680,APC,Alexa 633,Alexa 647,Dyomics 647,633nm Red laser trigon,530/30,502 LP,A,450/40,-,B,C,Alexa Fluor 430,DAPI,Hoechst,Alexa 405,Pacific Blue,407nm Violet laser trigon,