Methods in Cardiac Research 2016 Overview What is flow cytometry How does it work Applications Considerations for your project Questions What is it Laser based system that measures the properties of single cells ID: 1042316
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1. Flow cytometryAndreas RomaineMethods in Cardiac Research 2016
2. OverviewWhat is flow cytometry?How does it work?ApplicationsConsiderations for your projectQuestions?
3. What is it?Laser based system that measures the properties of single cellsMeasures the size, granularity and fluorescence intensity of cells and particlesCan compare the expression of multiple cell markers on individual cells
4. Flow cytometersBecton-Dickinson Fluorescence activated cell sorter (FACSCalibur)Becton-Dickinson ACCURI C6
5. How it worksLight scatter detection (antibody independent)Fluorescence detectionCombining parameters and GatingMulticolour flow cytometry and Compensation
6. Laser light sourceScattered light DetectorForward and Side ScatterForward light scatter (FSC)- sizeSide light scatter (SSC)- granularitySheath fluid flow
7. Forward and Side ScatterCell populations can be distinguished based on size and granularity
8. Antibodies and ControlsAntibodies are raised in a host species against single (monoclonal) or multiple (polyclonal) epitopesAntibodies can have varied specificities and affinitiesIsotype controls are antibodies raised in the same host species but with irrelevant epitope specificity Constant region Variable regionSheep anti mouse protein AConstant regionVariable regionSheep anti mouse protein B
9. Laser light sourceFluorescence DetectorFluorescence detectionFluorophoreAntibody
10. Mean fluorescence intensityFluorescence detectionFluorescence intensity is proportional to the expression of the protein/particle of interestCells can be fixed and permeabilized to assess intracellular expressionExtracellular and intracellular expression can be distinguished by quenching (uptake experiments)
11. Fluorescence detectionExcitation (Ex)The wavelength the flurochromes are excited byBlue laser 488nm or Red laser 640nm
12. GatingGates are used to select cells of interest, based on FSC/SSC or fluorescence or even a combinationReverse gating
13. Direct vs Indirect staining1° conjugated abs 2° conjugated abs Pros Cons FasterFewer wash steps= more cellsCan be more expensiveLimited fluorochrome selectionNot always availablelarger selection of fluorochromes that can avoid compensation issuesAmplifies signalAvoiding species cross reactivityMore wash steps1° ab2° conjugated ab
14. CompensationFL1FL2FL3Bleeding of FL1 signal into FL2 channel
15. Non transfectedPlasmid GFPAdenovirus GFP 41% 100%ApplicationsQuantification of transfection efficiencies
16. Results show 55.8% of the live cells are SDC4+Itga11-, whilst 31.9% of cells are double positive for SDC4 and Itga11Gates are based on isotype controls or secondary antibody only controlsApplications
17. ApplicationsCell sortingColocalisation- BRET
18. Cellular localizationWestern blottingFACSConfocal microscopyThroughputSample storageSmall samplesSimultaneous staininghighhighlowv. poorokv. goodLong termLostShort termNoYesYesNoYesYes
19. ConsiderationsCheck the specificity of your antibody first Fluorochrome selection be aware of the Ex. available on your cytometer and of spectral overlaps in Em.Choose brightest fluorochromes for lowest expressing target proteinsCross reactivity of secondary antibodiesKeep the cells alive or fix, but be consistentInclude controls with every capture