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Kumc introduction to flow cytometry

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Overview of flow cytometry

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Kumc introduction to flow cytometry

  1. 1. Introduction to Flow Cytometry Rich Hastings KUMC Flow Cytometry Core Lab 3901 Rainbow Boulevard Kansas City, KS 66160 913-588-0627 rhastings@kumc.edu http://www.kumc.edu/flow/
  2. 2. Flow Cytometry Flow (noun) = the motion characteristics of fluids. Cytometry (noun) = is a general name for a group of biological methods used to measure various parameters of cells. Parameters which can be measured by cytometric methods are cell size, the stage of the cell cycle, the DNA content of the cell, the existence or absence of specific proteins on the cell surface or in the cytoplasm, to name but a few. (Wikipedia)
  3. 3. Flow Cytometry All forms of Cytometry depend on the basic laws of physics, including those of fluidics, optics, and electronics. Watson, J. V. Cytometry, 38 2-14, 1999.
  4. 4. Flow Cytometry Flow cytometry is a system for sensing individual cells in a physiologic saline solution as they move in a focused liquid stream through a fixed laser beam scattering light and emitting fluorescence that is measured and converted into digitized data.
  5. 5. The History of Flow Cytometry 1930’s Moldavan in Science described the counting of blood cells filing through a capillary tube using a photoelectric sensor. 1940’s US Army constructed a device that detected bacteria in a stream of air, using a Ford headlight as a light source and a photomultiplier tube as a detector. 60% ~ 0.6 μm 1950’s Caspersson measured nucleic acid and protein metabolism for normal and abnormal cell growth using a Cadmium spark as a UV light source. Coulter constructed a cell counter based on the fact that the electrical conductivity of cells is lower than that of saline. Saline conducts, cells impede. 1960’s Kamentsky at IBM developed the Rapid Cell Spectrophotometer. It used an arc lamp source and measured nucleic acid content and cell size. A computer (!!!!) measured and analyzed the data. IBM loaned Herzenberg a prototype and he developed the first real FACS. 1970’s Industry takes over technical development: BD, Beckman, Ortho etc.
  6. 6. The History of Flow Cytometry Frequency distribution of DNA content Cells from a normal cervix Cells from a cervical carcinoma Premalignant cells from the epithelium Quantitative cytochemical studies on normal, malignant, premalignant and atypical cell populations from the human uterine cervix, Acta Cytologica 8, 1964 O. Caspersson 1964 Images from Dr. Louis Kamentsky
  7. 7. The History of Flow Cytometry The Coulter Counter: Counts cells and measures their size based. Patent The first commercial version of the Coulter Counter
  8. 8. The History of Flow Cytometry LA Kamentsky, MR Melamed & H. Derman, Spectrophotometer: New instrument for ultrarapid cell analysis, Science 150, 1965
  9. 9. The History of Flow Cytometry Herzenberg Lab at Stanford, Early sorters and analyzers.
  10. 10. The History of Flow Cytometry Flow cytometry has benefited from the technological development of: Monoclonal antibodies Fluorochromes DNA, RNA and Functional stains Computers and the miniaturization of Electronics Lasers
  11. 11. How Flow Cytometers Work The principal of hydrodynamic focusing confines cells to the core (sample) stream by a cell-free sheath fluid. Injector Tip Sheath fluid (PBS) Laser interrogates Stream Objective is to have one cell pass through the laser intercept at a time. What makes flow cytometry so powerful is the ability to gather data on each cell individually but having the capability to analyze 1000’s of cells/sec.
  12. 12. How Flow Cytometers Work Sheath pressure is constant. Sample pressure is variable. Aria LSR II Flow is laminar. Flow cell shape allows for hydrodynamic focusing. Hydrodynamic focusing causes cells to line up along their long axes.
  13. 13. How Flow Cytometers Work Higher Flow Rate = Good for Qualitative Measurements Lower Flow Rate = Greater Resolution and Quantitative Measurements From BD LSR II manual
  14. 14. How Flow Cytometers Work Laser is an acronym for Light Amplification by Stimulated Emission of Radiation 1. Lasers provide light of a specific wavelength. Most lasers used in flow cytometry operate in the visible spectrum. 2. An important aspect of laser light is coherence. Coherent light has the same wavelength, phase and direction. 3. Modern lasers generate light that is reliable and constant.
  15. 15. How Flow Cytometers Work Electromagnetic Spectrum Violet Laser Blue Laser Red Laser From: http://www.antonine-education.co.uk
  16. 16. How Flow Cytometers Work Laser interrogation of a cell tells us physical properties of that cell. Laser interrogates Cell in Flow Cell Incident light scattered at small angles (0.5- 2.0º) is called Forward Scatter (FSC) Incident light scattered at an angle of 90º is called Side Scatter (SSC)
  17. 17. How Flow Cytometers Work Forward Scatter (FSC) Rough measure of size, influenced by the wavelength of light, and the angle, lenses and apertures that light is collected at and with. Different flow cytometers will give slightly different FSC measurements. Most flow cytometers measure FSC with a photodiode. Bacteria – Photomultiplier tube (PMT) Dead cells may have lower FSC measurements than live cells. Osmotic swelling can increase cell volume, and decrease light scatter. FACSCalibur Epics XL Data: Shapiro and Becker
  18. 18. How Flow Cytometers Work Forward Scatter (FSC) “A big problem in the published literature is the use of forward scatter as a trigger/discriminator. Whilst fairly robust for leukocyte detection it is the most variable signal between systems and it is most alignment critical. It is affected by refractive index mismatches between sheath and sample, beam geometry, polarization, beam stop position, and collection angle. In some cases the relative forward scatter position of particles of different sizes does not follow their relative order in physical size. In jet-in-air sorters the beam geometry and the jet undulation at the intercept are critical factors whereas in cuvette-based instruments these tend to be dirt on the optical surfaces and slight rotation of the flow cell to the beam axis.” Gerhard Nebe-von-Caron Cytometry Part A 75A: 8689, 2009
  19. 19. How Flow Cytometers Work Side Scatter (SSC) is the measure of light scattered at an angle of 90º (orthogonal). SSC is a measure of the complexity of the cell’s internal structures. The more ‘granular’ a cell is the higher its SSC will be. A neutrophil is much more granular than a lymphocyte. Measured using a photomultiplier tube.
  20. 20. How Flow Cytometers Work FSC vs. SSC alone tells us a great deal about our cells. Lymphocyte Eosinophil Basophil Neutrophil Monocyte
  21. 21. How Flow Cytometers Work Pulse Processing From BD LSRII Manual Laser
  22. 22. How Flow Cytometers Work Parameters Measured Height is the maximum digitized intensity measured for the pulse. Area is the sum of all pulse heights. Width is Area ÷ Height x 64,000. From BD LSRII Manual
  23. 23. How Flow Cytometers Work Flow cytometry takes advantage of molecules fluorescing after excitation by laser light. Fluorochromes can be conjugated to monoclonal antibodies. Many functional stains exist that fluoresce. Energy acquired by the absorbance of light of certain wavelengths in these molecules drive electrons to a higher energy state in an unoccupied orbital. The return of the electron to ground state results in the emission of photons of longer wavelength (lower energy).
  24. 24. How Flow Cytometers Work Fluorochromes absorb energy (light) and their electrons go from a ground state to an excited state. The electrons return to ground state by emitting light of lower energy, therefore longer wavelength. Figures from Flow Cytometry - A Basic Introduction by Michael G. Ormerod
  25. 25. How Flow Cytometers Work Tandem Dyes – One molecule is excited by laser light and donates the energy to the acceptor molecule. Figure from Jane Limer BD Application Scientist
  26. 26. How Flow Cytometers Work Fluorochromes Synthetic, Organic Dyes: FITC, the Dylights (Thermo Fisher Scientific), Cy dyes, Alexa Fluors (Molecular Probes), Horizon dyes (Becton Dickinson), eFluor dyes (eBioscience), Pacific Blue, Krome Orange (Beckman Coulter), Brilliant Violet (Biolegend) Proteins: R-phycoerythrin (PE) is a photosynthetic pigment found in red algae. It is a 240 kDa protein with 23 phycoerythrobilin chromophores per molecule. Very bright, and excellent as the donator molecule in a tandem fluorophore. Allophycocyanin (APC) is a photosynthetic pigment found in bluegreen algae. APC is 105 kDa and has six phycocyanobilin chromophores per molecule. Very bright, and excellent as the donator molecule in a tandem fluorophore. Green Fluorescent Protein is “genetically encoded fluorescence” encoded by a single gene. A whole family of fluorescent proteins has originated from GFP.
  27. 27. How Flow Cytometers Work The photons emitted by excited fluorophores are routed to Photo- Multiplier Tubes (PMT). 1. Voltage is applied to the PMT making electrons present for the absorption of light energy from photons. 2. As more photons are detected, more electrons are recruited yielding a greater current on the detector. 3. IMPORTANT! If the PMT voltage is increased the same number of absorbed photons will have a greater current output, increasing the sensitivity of the PMT.
  28. 28. How Flow Cytometers Work The fluorescence intensity measured is proportional to the number of fluorescent molecules bound to the cell. From Applied Cytometry
  29. 29. How Flow Cytometers Work The Stokes Shift is the difference between the emission and excitation wavelength.
  30. 30. How Flow Cytometers Work Flow cytometers are engineered with precise light pathways. Light is routed through three different types of Filters. From BD LSR II manual
  31. 31. How Flow Cytometers Work Flow cytometers are engineered with precise light pathways. Cell Forward light scatter 0º A measure of cell size. 488 nm Side scatter 90º A measure of cell granularity.
  32. 32. How Flow Cytometers Work Flow cytometers are engineered with precise light pathways. 488 nm Light path in the LSRII
  33. 33. How Flow Cytometers Work 405 nm Violet Laser Bandpass Filter Fluorochromes 440/40 nm DAPI, Horizon V450, Alexa 405, Pacific Blue, Brilliant Violet 421 525/50 nm Pacific Orange, Cascade Yellow, Horizon V500, Brilliant Violet 570
  34. 34. How Flow Cytometers Work 488 nm Blue Laser Bandpass Filter Fluorochromes 780/60 nm PE-Cy7 695/40 nm PE/Cy5, PE/Cy5.5, PerCP, PerCP-Cy5.5 610/20 nm PI, PE-Texas Red 575/26 nm PE, Cy3 530/30 nm GFP, FITC, Alexa Fluor 488, CFSE
  35. 35. How Flow Cytometers Work 633 nm Red Laser Bandpass Filter Fluorochromes 780/60 nm APC/Cy7 710/50 nm Alexa Fluor 700 660/20 nm APC, Cy5, Alexa Fluor 647
  36. 36. Visualizing Flow Cytometry Data Dot plot: One parameter vs. another. Contour plot: One parameter vs. another showing the probability contouring. Density plot: One parameter vs. another, very good for viewing the frequency of subpopulations. Histogram plot: One parameter only. Y-axis is the count. X-axis is fluorescence intensity.
  37. 37. Visualizing Flow Cytometry Data Flow cytometry data: Iterative, Derivative, Visual, as well as Statistically Powerful.
  38. 38. Gating Gate on your Cells of Interest, The Population Hierarchy is your Friend.
  39. 39. Gating Gate on your Cells of Interest, Where are your Dead Cells?
  40. 40. Fluorescence Minus One (FMO) Unstained and Isotype Controls vs. FMO PE FMO Absence and Presence of CD4 PE Improper Compensation From FlowJo website/Mario Roederer
  41. 41. Doublets Doublet Discrimination: Two cells passing through the laser intercept concurrently. Doublet Discrimination: Doublets have double the area and width values of single cells.
  42. 42. Doublet Discrimination – Cell Cycle Voltage Intensity G0 G0 G0 G2/M 2N Height 2N + 2N Height 4N Height 2N Width 2N + 2N Width Stained nuclei separated by cytoplasm 4N Width Time
  43. 43. Doublet Discrimination – Cell Cycle Blue cells are the G0/G1 doublets. They have double the area and width values of single cells but lower height values than the G2/M cells.
  44. 44. Doublet Discrimination – Whole Cells A Good Tool for Gating: Doublets have Double the Width Value while maintaining Same Height Value. Blue Cells are singlets, Red Cells are doublets.
  45. 45. Compensation Compensation/Spectral Overlap Most fluorochromes and dyes excited by laser light have long emission curves. Flow cytometers have filter sets optimized for specific wavelengths of light. Unfortunately, overlapping emission wavelengths from one fluorochrome may spillover into the filter of another.
  46. 46. Compensation Visualizing Spectral Overlap
  47. 47. Compensation Compensation Controls  Compensation is very important. If one fluorochrome leaks into another’s channel, your data cannot be interpreted properly.  Each experiment needs an unstained control and each fluorochrome singularly. As long as your run the proper controls, we can determine the correct compensation values post acquisition.  Beads can be substituted for cells if cell number is a limiting factor. Spherotech, Invitrogen and Becton Dickinson offer anti-Ig beads that will bind fluorescently-labeled antibodies.
  48. 48. Compensation The FITC signal is leaking into the PE detector. We can adjust the PE-%FITC spectral overlap value until the unstained and FITC have the same mean value for PE. From BD LSR II manual
  49. 49. Compensation This figure is the Diva Compensation layout for a seven color experiment. FACS DIVA has a module designed for computing compensation. Module requires an unstained control and each color individually. Choose colors, then gate on the cells by FSC vs. SSC, and follow the layout.
  50. 50. Compensation (Pattern Recognition) Uncompensated Data Compensated Data
  51. 51. Compensation Statistics for a Four Color Experiment Mean Fluorescence Intensity Values
  52. 52. Compensation Uncompensated Data Voltages PE = 538 PerCP = 791 APC = 690 Compensated Data Comp Matrix PerCP -%PE = 22.01 APC - %PE = 0.09 APC - %PerCP = 4.54 PerCP - %APC = 1.04
  53. 53. Compensation Uncompensated Data Voltages PE = 650 PerCP = 825 APC = 725 Compensated Data Comp Matrix PerCP -%PE = 7.06 APC - %PE = 0.04 APC - %PerCP = 4.67 PerCP - %APC = 0.96
  54. 54. FlowJo Compensation FlowJo is a third party analysis software from Treestar, Inc. FlowJo has a built in Compensation Matrix/Wizard that is powerful and intuitive. Need single color and unstained controls.
  55. 55. FlowJo Compensation
  56. 56. FlowJo Compensation
  57. 57. Compensation Annexin-FITC and Propidium iodide (PI) need to be compensated. This can be difficult depending on where the cells are in the stages of apoptosis. We recommend Annexin-APC and PI, less spectral overlap.
  58. 58. Flow Cytometry Statistics For Histograms: y-axis = Number of cells/channel x-axis = Fluorescence intensity of designated parameter For Dot Plots: y-axis and x-axis = Fluorescence intensity of designated parameters Flow Cytometry is a qualitative assay, flow results depict the characteristics of your sample. Samples are measured in a dimensionless unit termed Fluorescence Intensity.
  59. 59. Flow Cytometry Statistics (Diva) • Number of events - total number of events in the defined population. • Parent - name of the next population up in the hierarchy. • %Parent - number of events in the defined population divided by the number of events in the parent gate (next population up in the hierarchy), expressed as a percentage. • %Grandparent - number of events in the defined population divided by the number of events in the grandparent gate (two populations up in the hierarchy), expressed as a percentage. • %Total - number of events in the defined population divided by the total number of events in the tube (all events), expressed as a percentage.
  60. 60. Flow Cytometry Statistics (Diva) Mean - Average linear value for events in the defined population, defined as: where n = number of events in the population, and Xi is a value for a particular parameter, where i = 1 to n. Geometric mean - Logarithmic average of the events in the defined population. This mean is less sensitive to outliers than the regular mean. The geometric mean is defined as: where n = number of events in the population, and Xi is a value for a particular parameter, where i = 1 to n.
  61. 61. Flow Cytometry Statistics (Diva) Two measures are generally made of a distribution: intensity and spread. In flow cytometry, the intensity of a distribution can be represented by the position of the “center” of the distribution. The “center” is usually represented mathematically by the mean, median or peak channel number. If the data has been displayed on a linear scale, the arithmetic mean is used; for logarithmically displayed data, the geometric mean is generally chosen. If any part of the distribution lies off scale at either end of the axis, the value for the mean channel number will be inaccurate and should not be used; the median channel can be used as long as more than half of the distribution in on scale. Flow Cytometry - A Basic Introduction Michael G Ormerod
  62. 62. Flow Cytometry Statistics (Diva) The peak channel number is an inaccurate measure of the center of a distribution and is not recommended. For a Guassian (normal) distribution, these three values should be equal. The spread of a distribution is usually expressed as the Standard Deviation (SD). However, in flow cytometry, the coefficient of variation (CV) is preferred because it is dimensionless and, on a linear scale, does not depend on where in the histogram the data is recorded.(CV = SD/mean channel number). Flow Cytometry - A Basic Introduction Michael G Ormerod
  63. 63. Immunophenotyping Cell Staining Overview  Stain 105-106 cells/tube  Tubes vs. Plates  Stain cells in small volumes.  Titer antibodies.  Block Fc with species specific antibodies.  Direct vs. Indirect staining  Fixation
  64. 64. Immunophenotyping-Antibody Dilution Most antibody manufacturers advise a dilution to start with. We advise performing a dilution curve. Figure from Flow Cytometry - A Basic Introduction by Michael G. Ormerod
  65. 65. Immunophenotyping-Dilution Protocol 43.3%, Mean = 6982 37.3%, Mean = 4923 36.6%, Mean = 4958 32.4%, Mean = 3784 32.4%, Mean = 3886 2.14%, Mean = 1042 1.4%, Mean = 886 32.4%, Mean = 3886 2.14%, Mean = 1042 1.4%, Mean = 886
  66. 66. Immunophenotyping Gate on physical properties of cells. Then, gate on the live cells. 7AAD negative cells. Then, gate on CD3+, CD56- T-cells. Then, gate on CD4+ vs. CD8+.
  67. 67. Immunophenotyping Multi-color experiments – Overcome the Complexity
  68. 68. Setting up an Experiment From Lora Barsky, USC Flow Core
  69. 69. Immunophenotyping Multi-color experiments – Overcome the Complexity http://www.fluorish.com/ http://www.biolegend.com/panelselector http://www.ebioscience.com/resources/fluorplan-spectra- viewer.htm http://www.beckmancoulter.com/wsrportal/wsr/re search-and-discovery/products-and-services/ flow-cytometry/research-tools/ index.htm http://www.bdbiosciences.com/ecat/paneldesign er.jsp http://www.chromocyte.com/calculate http://www.invitrogen.com/site/us/en/home/supp ort/Research-Tools/Fluorescence- SpectraViewer.html
  70. 70. Cell Cycle  Figure from Purdue University Cytometry laboratories. G1 M G2 M (Mitosis) Dividing the replicated chromosomes. S G0 G1 (Gap1) Quiescent cells interval between mitosis and initiation of DNA replication, RNA polymerases have access to the genome, much protein synthesis. G2 (Gap2) interval between DNA replication and mitosis S interval of time in which the DNA is replicated
  71. 71. Cell Cycle From Becton Dickinson
  72. 72. Cell Cycle  DAPI – Excitation maximum = 358 nm, Emission maximum = 461 nm  DAPI is bound to dsDNA in AT clusters in the minor groove.  Because DAPI is excited by the violet laser and emits in the blue wavelengths, it is an excellent counter-stain for yellow, green and red fluorochromes.
  73. 73. Cell Cycle  Propidium Iodide – Excitation maximum = 493 nm, Emission maximum = 632 nm  PI is bound to DNA by intercalating between bases with no preference for purine or pyrimidine base pairs. PI will also bind to RNA.  One PI molecule per 4-5 base pairs.  PI cannot pass through intact cell membranes, cells need to be dying or permeabilized to allow PI staining.
  74. 74. Cell Cycle Doublet Discrimination is very important with this technique!!!
  75. 75. Apoptosis  Programmed Cell Death  Characterized by DNA fragmentation and distinct changes in cell morphology and volume.  Requires biochemical energy.  Important – For the normal functioning of the immune system, embryonic development, normal tissue maintenance and chemical- and hormone-induced cell death.  ‘Programmed’-the genetically determined eradication of cells.  Part of normal cell development, aging, and as a security mechanism.  Necessary and Pathological.
  76. 76. Apoptosis vs. Necrosis  Necrosis  Toxicity-induced cell death.  Requires no energy, passive.  Cells swell and then karyolysis (dissolution of the chromatin and nucleus - DNase).  Release of cellular contents may cause inflammation.  Apoptosis  ‘Stimulation’-induced cell death.  Energy required.  Cell shrinkage, then pyknosis (chromatin condenses), followed by karyorrhexis (fragmentation of the nucleus).  Do not release cellular contents and are readily phagocytosed by macrophages.
  77. 77. Apoptosis  Apoptotic Effects - Cell Morphology  Cells change shape and shrink during apoptosis.  The chromatin condenses in a process called Pyknosis.  The cells become smaller and the cytoplasm shrinks around the organelles. Figure from the Cell Migration Lab, University of Reading http://www.reading.ac.uk/c ellmigration/apoptosis.htm
  78. 78. TUNEL Assay  TUNEL (Terminal dUTP Nick-End Labeling)  During Apoptosis, Genomic DNA is cleaved into small double-stranded fragments and single-stranded breaks called ‘nicks’.  Terminal deoxynucleotidyl transferase (TdT) labels DNA strand breaks by catalyzing the polymerization of labeled nucleotides to free 3’-OH DNA ends.  The 3′-OH ends of the breaks can be detected by attaching a fluorochrome. This is generally done directly or indirectly (biotin) using fluorochrome-labeled deoxynucleotides in a reaction catalyzed preferably by TdT.  Best results are achieved using a positive control (fixed, permeabilized cells treated with Dnase) and a negative control (no FITC labeling reagent).  We have had good luck with the Roche kit (cat # 11 684 795 910).
  79. 79. TUNEL Assay Gated on Sperm Negative Control Tunel FITC, No PI Positive Control No Tunel, PI Only Positive Control Tunel FITC and PI Positive Control Tunel FITC and PI Test
  80. 80. Annexin V Assay  Timeline  1990 Andree at al. found that a protein, Vascular Anticoagulant , bound to phospholipid bilayers in a calcium dependent manner. Protein was renamed Annexin V.  1992 Fadok et al. discovered that macrophages specifically recognize phospatidylserine (PS) that is exposed on the surface of lymphocytes during the development of apoptosis. This PS is normally on the inner leaflet of the membrane.  1994 Koopman et al. developed a flow cytometric assay for measuring FITC conjugated Annexin V binding to apoptotic cells. Stained control and serum starved cells with ethidium bromide and Annexin V-FITC.
  81. 81. Annexin V Assay = Phosphatidylserine Normal Cell Membrane No PS on surface. Apoptotic Cell Membrane PS on surface. Apoptotic/Necrotic Cell Membrane PS on surface, membrane disintegrates.
  82. 82. Annexin V + PI Apoptosis Assay Annexin V binds to Phosphatidylserine on the Cell Membrane, PI to DNA
  83. 83. Cell Sorting
  84. 84. Flow Cytometry Assays Immunophenotyping DNA cell cycle/tumor ploidy Cell tracking and proliferation Cell Viability, Apoptosis, Necrosis Fluorescent Protein expression Cell Sorting Cell Counting and Antigen quantification Membrane and mitochondrial membrane potential Intracellular protein staining pH changes - BCECF Redox state - NADH Chromatin structure – Acridine Orange or 7-AAD Total protein – Low MW dyes that bind to charged groups on proteins Lipids – Nile Red Surface charge – Fluoresceinated polycations Membrane fusion/runover – MC540 Enzyme activity – Caspase, lysosomal, kinases etc Sulfhydryl groups/glutathione – Oxidative metabolism, Fluorescien-5- maleimide DNA synthesis – Mitotic index DNA degradation – apoptosis-associated DNA degradation Gene expression RNA Content – Pyronin Y Cell Activation

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