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Applications in Flow Cytometry

Applications in Flow Cytometry. Cláudia Bispo. IGC – April 4, 2013. Outline. Potential Applications of Flow Cytometry. Cell State. Immunophenotyping Cell cycle Apoptosis Cell proliferation. Cell Function. Cell activation Calcium flux Cytokine Secretion

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Applications in Flow Cytometry

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  1. Applications in Flow Cytometry Cláudia Bispo IGC – April 4, 2013

  2. Outline Potential Applications of Flow Cytometry CellState • Immunophenotyping • Cellcycle • Apoptosis • Cell proliferation CellFunction • Cell activation • Calcium flux • Cytokine Secretion • Activation of signalling pathways • Levels of intracellular reactive oxygen species Microbiology • Dead/Live Discrimination • Absolute counting • Time points

  3. EvaluateCellState

  4. Immunophenotyping • Uses labeledantibodies (Abs) to identifycellsofinterest • Determination of cell surface antigens • Allows for detailed identification of cellular subsets (simultaneously measure multiple parameters cell by cell) • Targets on both surface and intracellularly CAUTION – Absselection: Fluorophore’sexcitationspectrum must match the laser lineused, anditsemission must fallwithindetectionfilter sets in thecytometer

  5. CellCycle DNA content analysis - PropidiumIodide (PI) M G0 G0/G1 G1 G2 G2/M S S-phase Fluorescence (DNA content) Excitation / Emission : 488nm / max 617nm

  6. CellCycleAnalysis Cell Cycle Analysis Software G0/G1 Cell Number Examples: FlowJo ModFit LT  FCS Express  IDLYK … G2/M S Fluorescence Intensity • Accurate measurements allow for resolution of normal cells undergoing G1, S, G2 phases • Also useful when multiple DNA populationspresent: measuring aneuploidy & polyploidy

  7. CellCycle- Bromodeoxyuridine (BrdU) method PropidiumIodide plusBrdU staining • BrdU is thymidine analog • Taken up by cells in S-phase • Usually in combination with PI 104 S Phase 103 102 Anti-BrdU FITC 101 G1 G2/M Excitation / Emission : PI 488nm / max 617nm BrdU varies byfluorophore Propidium Iodide

  8. CellCycle - G0/G1 discrimination Pyronin Y plusHoechst 33342/33258 G0/G1 G0 G1 S G2/M S CellCount G2/M RNA content RNA Content DNA content (A-T base pairs) Excitation / Emission : PY 488nm/ 575nm HO UV line / 460-490nm

  9. Apoptosis Changes in light scatter DNA denaturation Changes in plasma membrane Changes in cell organelles / signaling pathways * positive control is useful

  10. Apoptosis CELL DEATH – FSC x SSC Changes in light scatter: low level resolution of apoptotic cells

  11. Apoptosis Propidium Iodide (fixed cells) DNA Degradation • Other viability dyes : • 7-AAD • Zombie Aqua • To‐Pro3 • ...

  12. Apoptosis Annexin V-fluorochromeplusPropidium Iodide (non-fixed cells)

  13. Apoptosis Annexin V plusPropidiumIodide Excitation / Emission : Annexin varies byfluorophore PI 488nm / 617nm

  14. Add Antibody Analysis by Flow Cytometry Apoptosis (intracellularstaining) Fix and permeabilize

  15. Apoptosis – Bcl-2 familymembers Excitation / Emission: Varies byfluorophore

  16. Apoptosis – ActivatedformsofCaspases Untreated Etoposide FlowcytometricanalysisofJurkatcells, untreated (blue) oretoposide-treated (green), usingCleaved Caspase-3(Asp175) Antibody (AlexaFluor® 488 Conjugate). Excitation / Emission: Varies byfluorophore Ex A488: 488nm / 520nm

  17. Dilution of CFSE Cell Divisions CellProliferation • Tracking Cell Proliferation with CFSE (Carboxyfluorescein Succinimidyl ester) STAIN WITH CFSE CELL 3 1 2 4 0 Excitation / Emission: 488nm / 521 nm

  18. IL-7 IL-7+ DMSO IL-7+ ErkInhibitor IL-7+ PI3K Inhibitor TrackingCellProliferationwith CFSE

  19. EvaluateCellFunction

  20. CellActivation FSC x SSC – Cell size Sofia Marques (IMM) Filipa Lopes (IMM)

  21. CellActivation Activation markers: CD69, CD71, etc Excitation / Emission: Varies byfluorophore

  22. CalciumFlux Effects of T cell receptor stimulation by B-Cells on ionized calcium concentration ([Ca2+]i). Fluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophorFluo-4 Excitation / Emission: Indo1 UV line / 405nm Fluo-4 488nm / 516nm Sofia Marques (IMM) Filipa Lopes (IMM)

  23. Activationofsignallingpathways Krutziket al. ClinImmunology (2004)

  24. pStat1 Activationofsignallingpathways Phospho-protein detection • Ability to analyze rare subsets of cells within complex populations • Better evaluate population heterogeneity • Discrimination of High vs. Low responders Krutziket al. ClinImmunology (2004)

  25. CombiningSurfaceMarkerswithPhospho-staining  Evaluate protein phosphorylation in different subpopulations concurrently

  26. Levels of intracellular reactive oxygen species Excitation / Emission: 488nm / 605nm Measurement of oxidative burst in human peripheral blood granulocytes using dihydroethidium. On oxidation, ethidium is produced which binds to DNA & fluoresces red. Thehistogram shows theredfluorescencebeforeandafterincubationwith 10 mMmuramyldipeptide for 15 min.

  27. CytokineSecretion - Multiplex BeadArrays SOON @ UIC Luminex MAGPix System bead coated with capture antibody mixture Quantitation & detection of cytokine and signal transduction proteins, DNA & RNA - Magneticbead-basedtechnology - Up to 50 analytes /well of a 96 well plate - Up to 4.800 results in 1 hour - Uses small sample volumes

  28. MultiplexBeadArrays SOON @ UIC Or design custom and personalized panels Merck Millipore MAGPix

  29. MicrobiologyApplications

  30. Microbiology - Dead/Live Staining • Various established methologies can be optimized • SYTO 9 /PI - Live/Dead BacLight viability kit • SYBR Green-I /PI - Barbesti (2000) • DiBAC4(3)/EB/PI - Flow Cytometry Protocols 2nd ed. • etc... • Several kits available Excitation / Emission: 488nm / Varies bymethod LIVE/DEAD kit for Bacteria LIVE/DEAD kit for Yeast & Fungi CAUTION: Make sure excitation/emission of kit dyes do not coincide with the ones on your sample

  31. Microbiology MonitoringCellCycle – DNA content Minor protocoladjustments/optimization might be needed comparatively with established protocols for eukaryotes E.coli: DRAQ5 - Silva et al. (2010)Yeast: PI or SYTOX Green - Knutsen (2011) Excitation / Emission: 647nm/ 670nm Excitation / Emission: 488nm/ 520nm

  32. Microbiology Time Points Assess treatment effect on population survival, protein expression, etc at indicated time points - effect on protein expression - counting: population proportions CAUTION: Make sure you use a fluorescence controlwhenprotocolrequires measuring & comparing protein expression

  33. Cellcounting Beads • Employs the use of reference beads on a regular cytometer • Allows the examination of large number of cells per sample • Combining Surface Markers will allow to count specific subpopulations @ UIC Scepter • Handheld Automated Cell Counter • Allows cell differentiation by its volume (size) according to the Coulter principle • Uses a disposable sensor

  34. Cell counting (2) @ UIC SOON @ UIC Imagebasedalternatives • Countess™ Automated Cell Counter(Invitrogen) • Tali™Image-BasedCytometer (Invitrogen) • TC20 AutomatedCellCounter (BioRad) • etc...

  35. Today’s Future Applications

  36. Amnis Image Stream Merck Millipore

  37. Amnis Image Stream

  38. CyTOF – Mass Cytometer • Can measure 34 parameters simultaneously in a single cell • Cells are labeled with Abs containing rare elements metal tags • Cells are atomized & ionized in plasma (high temperatures) • Tags are separated & identified by mass (time-of-flight) Omatsky et al. JAAS (2008), Bendall et al. (2011)

  39. Future Advances • Heading further into the path of Single Cell Analysis • microfluidics& lab-on-a-chip systems • Reduced laser size and capillary flow techniques mean smaller instruments • Instruments can now image cell at point of laser interrogation • More colours for immunofluorescence

  40. SummaryFlowLabEquipment@ UIC

  41. Summaryofapplications @ UIC FlowLab

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