Principles of Flow Cytometry

1、Orifice = 50 to 400 Principles of Flow CytometryQuartznozzleFluorescence signalsFocalized laser beamInjection of cellsFluorescencePhotodiodeLight can be measured at 90 : Side scatter + FluorescenceSide scatter reflects the cell contentLaserFluorescence intensityFITCFITC101104103102Relative fluoresce

2、nce intensityNumber of EventsFITCFITCFITCFITCFITCFITCFITCFITCBasics of Flow CytometryCells in suspensionflow in single-file throughan illuminated volume where theyscatter light and emit fluorescencethat is collected, filtered andconverted to digital valuesthat are stored on a computerFluidicsOpticsE

3、lectronicsThe automated MicroscopeWasteDetector& CounterSampleThis 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!1Hydrodynamic focussing in the cuvetteSh

4、eathSampleSheathSampleSample pressure low, small core stream. Good for DNA analysisHigh sample pressure, broader core stream.Bad for DNA analysisLOWHIGH Pressure (= Sheath Pressure) drives the sheath buffer through the cuvette, and the higher pressure in the sample tube(= Sample Differential) delive

5、rs 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

6、cell suspensionsSummaryBasic opticscA system of prisms and lenses directs the laser light to the interrogation point in the cuvetteLaser delaySheathSampleUmouje cross beam kompenzaciVyaduje stabiln fluidicsSummaryExcitation light is steered with prisms and lenses to the interception point Emitted li

7、ght is collected using lenses and is split up with dichroic mirrors and filters Tasks for the electronical systemConvert the optical signals into electonic signals (voltage pulses) Digitise the dataAnalyse Height (H), Width (W) and Area (A) of the pulseSend the data to the analysis computerHow a vol

8、tage pulse from the PMT is generated VoltageLaserLaserLasertttVoltageVoltage1.2.3.Height, Area, and WidthTime (s)VoltagePulse area(A)Pulse Height (H)Pulse Width (W)400ThresholdThe threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower

9、intensity are not displayed and not recorded for later analysis.SummaryDuring passing the laser voltage pulses are generated at the PMTAmplifiers enhance the signalsOnly signals passing the desired threshold(s) are analysed and recorded The data are finally passed to the analysis computer connected

10、to the cytometerAn overviewYearInstrumentIntroducedMost FrequentlyHeard Comments1976FACS IIWhen can I get one?1991FACS VantageDo you really need 5 colors?1998FACS Vantage SEDo you really need 6 colors?2000FACS DiVaDo you really need 8 colors?1980FACS IV/440Do you really need 4 colors?2003FACS AriaWh

11、en 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) Apoptos

12、is (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 ExpressionWhat are the advantages / disadvantages? Advantages Save Time and Samples (1) 6-color stain

13、 = (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

14、 errors in compensation Proper controls requiredExcitation- and Emission spectra of dyes for the blue laserStejn excitace rzn emisePekryv spekter(overlap)Excitace jinm laserem?Compensation bdbiosciences /spectra / How much compensation is correct?PEPEImportance of ACCURATE CompensationRPCILFCn = neg

15、ativesd = dim positivesb = bright positivesPE-CY5-CD8APC CD45ndbndbUncompensatedCompensatedOver CompensatedbnWhere 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 bri

16、ght markers to setup proper compensation.Hardware Compensation How to set compensation on the instrumentSetting compensationPrepare 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.Alig

17、n 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 VoltageFL1-no stainFL2-no stainUncompensatedCompensatedMedian value

18、s both = 3.2 Setting compensation - FITC single stainFL1-FITC CD3FL2-no stainFL2-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 CD3Setting compensation - PE singl

19、e 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. CompensatedMedian values both = 2.5 FL2-PE CD4FL1-no stainCompensation Controls Single Stain ControlsDoes not matter as long as

20、: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

21、compensation is specific for the fluorochrome, not the cell typeSingle Stain Controls - Which cells?Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sampleORA different antibody may be substituted, as long as it is conjugated to the same fluorochrome.HoweverSingle Stain C

22、ontrols - which reagents?Caveats for substituting reagents:Controls should be as bright as possibleAs bright or brighter than the experimental stainsGFP, 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 s

23、ame reagent spillover varies from reagent to reagent Single Stain Controls - which reagents?Compensation of tandem-conjugates can differ from lot to lotUse same reagent as experimental sampleLots positiveSmall CV, brightSome reagents wont work (IgL, EMA/PI)can mix with regular compsUsing Antibody Ca

24、pture Beadsas single stained controlsSoftware Compensation Automated Tools for Setting CompensationCompensation ToolsMust have single stained controlsSoftware calculated compensation for you!Easy, accurate and quick.Makes MULTI- Color compensation possibleSoftware Compensation ToolsAvailable on new

25、generation machinesDakoCytomations Summit (version 4)Coulter FC500 BD DivaOthersPost-acquisition softwareFCS ExpressFCS PressWinList FlowJoOthers Compensation MatrixFL1FL2FL3FL1 Comp3.960FL2 Comp27.355.15FL3 Comp011.18Compensation - Automatic MethodAutomatic compensation in the Diva software offers

26、a fast, easy and reliable method to set the correct compensation. First, select Create Compensation Tubes“ from the Instrument Menu:Compensation - Automatic MethodThe software automatically creates a list of single color tubes, based on your instrument setting.Naturally, in certain experiments you m

27、ay 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 LessonsProper CONTROLS are essentialDONT compensate by eyeUse Median to adju

28、st the populations if you must do it manuallyTRUST the software to do it for youIt does it quicker and more accuratelyPolychromatick cytometrieDesign experimentu a analzaChildhood Leukemia Investigation Prague -stav imunologie, Klinika dtsk hematologie a onkologie, UK 2.LF a FN MotolPrahaWhich fluor

29、ochrome 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 con

30、jugates Single or multiple laserComparison of the dye intensity for the same markerBaumgarth, Roederer, JIM, 2000, A practical approach to multicolor flow cytometry for immunophenotypingSpektra fluorochrom bdbiosciences /spectra / Which fluorochrome for which marker? In general, try to use brighter

31、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 conjugate

32、s (FITC or PerCP) for antigens expressed at high levels (e.g. B220 or CD4)Zkreslen vlivem kompenzacPEFITCPEPE-TxRedPesvit (spilover, spectral overlap)z PE do FITC je mal = mal kompenzacez PE do PE-TxRed je velk = velk komp.PE-TxRed PE = 65%Grafick eenLoglinear transformation“Biexponencial display“Zkreslen vlivem kompenzacJe teba promyslet odkud se dvatObvykl problmy: PE vs PE-TxRed, PE-Cy5 vs APCNelze pout vdy histogram Nelze vdy pout tverce i kvadrantyJe teba promyslet jak postavit gate (kontroly FMO)V siln komp. kanlech je men rozlien a hor kvantifikaceD