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Fundamentals and Applications of Flow Cytometry Scott Tighe Flow Cytometry Core Lab at the Vermont Cancer Center HSRF

Fundamentals and Applications of Flow Cytometry Scott Tighe Flow Cytometry Core Lab at the Vermont Cancer Center HSRF 305 656-2557. Basics components of a cytometer Fundamentals of photonics-optics and fluorescence Software and modeling Types of analysis

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Fundamentals and Applications of Flow Cytometry Scott Tighe Flow Cytometry Core Lab at the Vermont Cancer Center HSRF

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  1. Fundamentals and Applications of Flow Cytometry Scott TigheFlow Cytometry Core Lab at the Vermont Cancer Center HSRF 305 656-2557

  2. Basics components of a cytometer Fundamentals of photonics-optics and fluorescence Software and modeling Types of analysis Sorting for cells and RNA Sample requirements-[controls, compensation, titer Ab, blocking] Sign-up for time on VCC instruments Overview

  3. What is Flow Cytometry?An instrument for making cell-based fluorescent measurements.Method for quantitating cellular or structural components of a cell using fluorescent antibodies or probes. Allows Analysis of tens of thousands of cells in minutes.A method to sort and collect specific cell types. Beckman Coulter Epics XL The BD FACS ARIA with sorter

  4. Hardware Components of Flow CytometerFluidics- Cells are carried to laser in a saline-based sheath fluid.Pneumatics-Pressure drives the fluid flowOptics- laser, band-pass filters, andPMT detectors.Computer-Performs the analysis Older MoFlo cytometer by Cytomation

  5. THE FLOW CELL Laser focusing and hydrodynamic focusing LASER Sample Pressure SheathPressure (Constant) Waste Tank Sheath Tank Vacuum Line Pressure Fluidics

  6. Injector Tip Sheath fluid Fluorescence signals Focused laser beam The Flow cell:showing hydrodynamic focusing Higher the sample flow the wider the sample stream and lower the resolution

  7. Excitation Sources

  8. Lets consider two items Fluorochrome excitation-emission curve The electromagnetic spectrum

  9. Lasers provide Coherent Light (Single mode-single wavelength) High Power Narrow band width Can be tunable or fixed wavelength Gas tube or Pumped solid state-which allows coverage for all fluorochromes! GASPSS Krypton-647nm Nd-Yag (AlGaAs diode or Krypton lamp pumped) Argon -488nm Freq. Tripling !!! 1064,532,355nm HeNe [633nm] Great for Flow cytometry UV-364nm [from Ar] Ti:sapphire (Ar pumped) 650-1100 nm Excimers [193-284nm] Lasers This is just a few….

  10. Output frequencies of common tunable gas tube lasers

  11. -Benefits Cheaper Smaller Easily Available Many wavelengths Light Emitting Diodes [LED] -Cons -Not as bright -Broad excitation spectra -Not available for UV

  12. Fluorescence and Photons

  13. the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. Fluorescence Ext. coef + Quantum yield is Quantum efficiency Stoke shift Release of Photon

  14. The fluorochrome: Phycoerytherin 488 nm Argon 575 nm Large multi-subunit, globular (~240 kDa) Excitation 488 nm Emission Maximum 575 nm >20 chromophores per molecule High quantum yield (bright)

  15. Optics and Detection

  16. Green Signal Red Signal Detecting and sorting of different colors from cellular emission involves band pass filter and beam splitters Laser (Argon 488nm) FS Sensor Fluorescence detector (PMT1, PMT4 etc.)

  17. Optics in a Flow Cytometer Beam Splitters

  18. Forward Angle [FS] Light ScatterProvides data on size- The bigger the cell, the larger the FS Laser FS Sensor [size]

  19. Laser FS Sensor SS Sensor [granularity] Side Scatter [SS] DetectorProvides data on internal structures The more structures, the more ss Granulocytes have high ssProvides data on surface characteristics Dead cell have a rougher cell surface and a higher ss RBC have little to no ss

  20. Beam Splitters Dichroic Filter/Mirror at 45 degrees Laser Light+ Sample Light BS 625 Transmitted Light 500-680nm 488-680nm 625nm and above 500LP 500-624 reflected Reflected light

  21. Band Pass Filters 630/30nm BandPass Filter White Light Source Transmitted Light A 630/30 BPF will only allow 615-645nm through

  22. Long Pass Filters 520 nm Long Pass Filter Light Source >520 nm Light Note: Great for microscopes Short Pass Filters 575 nm Short Pass Filter Light Source <575 nm Light

  23. Detectors

  24. Photomultiplier tubes (PMT) Photodiode (PD) “Old” but good technology Most common detector used in flow High sensitivity but poor quantum efficiencies in red (>650nm) Able to adjust gain to over 100,000 Common Inexpensive New[er] technology, still not common High quantum efficiencies for visible No internal gain adjustment Requires Cooling Require a high voltage bias

  25. PMT 4 Bandpass filter PMT 3 Dichroic filters 2 PMT 1 PMT Laser Flow cytometers may have 4 to 6 detector channels [or more] Flow cell PMT=Photomultiplier Tubes-do not see colors, only photons.

  26. Spectral Compensation Must be performed when using fluorochromes with overlapping emissions

  27. Sorting Cells

  28. The Cytomation Mo Flo cell sorter Analyzes and sorts cells at 70,000 cells per second Cost $ 350,000

  29. Sorting + FS Sensor 488 nm laser Fluorescence detector - Charged Plates Single cells sorted into test tubes

  30. Data Output

  31. Data Output is represented by a histogram -Single parameter trace [one color] -Dual paramater dot plot [two color] Histograms Fluorescent intensity-

  32. Gating • Allows the ability to select specific cell populations in one histogram and analyze for additional parameters [colors] in additional histograms

  33. Comparison of single and dual parameter histograms

  34. Specific Types of Analysis Done Using Flow Cytometry

  35. Some Typical Applications of Flow Cytometry? • Immunophenotyping • DNA cell cycle/tumor ploidy • Membrane potential • Ion flux • Cell viability • Karyotyping • Cell tracking and proliferation • Sorting • Redox state • Chromatin structure Cell proliferation assay Cell enumeration and sizing Apoptosis Phagocytosis Intracellular pH Intracellular calcium Oxidative burst Intracellular antigen measurement Cytokine detection Reticulocyte analysis Platelet analysis

  36. Immunophenotyping

  37. CD # = cluster designation number ImmunophenotypingClassifying immune cells using cell surface antigens CD3 CD4 CD3-T-cell CD4-T helper

  38. The structure of IgG-An Antibody Fab regions Fab regions Fc receptor

  39. Two Types of Antibody Labeling Direct labeling: Uses one antibody that has a fluorochrome conjugated directly on it. One step staining. Easier. Indirect labeling: Uses two antibodies.The first is “against” a specific antigen on the cell. The second antibody is fluorochrome-labeled and is “against” the first. More complicated. Cell Cell

  40. Example of DataUsing two mABs with dyes of different color outputs .1 1 10 100 1000

  41. Non-Specific Antigen Blocking Blocking is important to avoid false positives Non-specific binding of antibodies is really Fc bindingTypically a serum source (BSA, FCS) is used but is often not adequate. Recommend goat IgG at 100-200ug/ml

  42. mAB Titering-A must!!!! Uses a specific Number of cells against antibody dilutions. Perform on new lots of antibodies

  43. Cell Cycle

  44. M G0 G2 G1 S Phase (Synthesis) s Count 0 200 400 600 800 1000 2N G1/G0 4N G2M Cell Cycle AnalysisIndicates the rate and stage of cell replication or division.Propidium Iodide most common dye.The dye intercalates into the DNA strand.

  45. Modeling Cell Cycle dataWhen peaks are close together and overlapping, it is important to use specific software to model the data and get accurate results ModFit 3D 3.0 WinList 5.0 FloJo WinMDI is a free software written by J.Trotter and is available on the web.

  46. Cell Cycle of GFP Cells

  47. Cell Viability

  48. Cell ViabilitySimplest method using cell permeabilization -Propidium Iodide -7AAD -SytoxMembrane potentials are a good indicator such mitochondria membrane potential [JC1]. Redox dyes-DHF, DHR- Turn colorless by reduction in cells.Enzyme activity probes-Esterase activity using cFDA, cell tracker dyes, calcein,… converted to fluorescent probe by enzymes.

  49. Cell Viability using Dye Exclusion-Propidium Iodide • How the assay works: • PI cannot normally cross the cell membrane • If the PI penetrates the cell membrane, it is assumed to be damaged • Cells that are brightly fluorescent with the PI are damaged or dead Dead Live PI PI Pi fluoresence >>> PI PI PI PI PI PI PI PI PI PI PI PI

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