Introduction to Flow Cytometry

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1 Outline Introduction to Flow Cytometry Basic Concept of Flow Cytometry Introduction to Instrument Subsystems Daisy Kuo Assistant Product Manager BDBiosciences Application Examples 2 What is Flow Cytometry? Flow = Fluid Cyto = Cell Metry = Measurement Particle Size Detection range: 0.5~50um A variety of measurements are made on cells, cell organelles, and other objects suspended in a liquid and flowing at rates of several thousands per second through a flow chamber. 3 4 What Can a Flow Cytometer Tell Us About a Cell? Its relative size (Forward Scatter FSC) Scatter Light Laser Its relative granularity or internal complexity (Side Scatter SSC) FSC Sensor Its relative fluorescence intensity SSC Sensor 5 6

2 Lysed Whole Blood Fluorescence Light Side Scatter Neutrophils Forward Scatter Monocytes Lymphocytes 7 8 Fluorescence Applications Phenotype Analysis (Cell Surface Antigens/Markers) Intracellular Analysis -- Eg. Cytokines, Signal Transduction molecules etc. DNA Analysis -- Eg. Viability, Cell cycle, Apoptosis etc. Cell Fuction Analysis -- Eg. Free radicals, Ca 2+, Reporter genes etc. CBA (Cytometric Bead Array) 9 10 BD Flow Cytometers Cell Analyzer BD Flow Cytometers Cell Sorter FACSCalibur FACSCanto FACSAria 2 Lasers, 4 Colors Analyzer 2 Lasers, 6 Colors Analyzer 11 3 Lasers, 11 Colors Sorter 12

3 Subsystems Fluidics Pressurized System Fluidics To introduce and focus the cells for interrogation. Optics To generate and collect the light signals. Electronics To convert the optical signals to proportional digital signals, process the signals, and communicate with the computer Flow Cell: Sample Flow Sample Differential Excitation Lasers Low Differential Pressure High Differential Pressure Hydrodynamic Focusing Low Sample Pressure 12 µl/min Laminar Flow High Sample Pressure 60 µl/min Laminar Flow sheath sample sheath Sheath Sample Sheath Sheath Sample Sheath Fluorochrome Spectra Optics Excitation optics Lasers: 488nm(Blue) + 633nm(Red) Lenses to shape and focus the laser beam Collection optics A collection lens to collect light emitted from the article-laser beam interaction A system of optical mirrors and filters to route specified wavelengths of emitted light to designated optical detectors 17 18

4 FACSCalibur Optics Optical Filters FACSCalibur Optics Collection Optics SSC 488/10 FL1 530/30 FACSCalibur Configuration FL2 585/42 90/10 Beam Splitter FL4 661/16 DM 560SP DM 640LP 670LP Half Mirror Fluorescence Collection Lens FL3 488 nm Blue Laser Excitation Optics ~635 nm Red Diode Laser Focusing Lens Flow Cell 488/10 FSC Diode FACSCanto Excitation Optics FACSCanto Collection Optics 633nm 488nm 23 24

5 Detector Blocks Octagon and Trigon Collection Optics Octagon Collection Optics Trigon Optics-- Configuration Electronics Creation of a Voltage Pulse PMTs and preamps convert photons to voltage pulses. Analog-to-digital converters translate analog signals to proportional digital signals. Laser Pulse Height Compute area and height for each pulse. Perform compensation and calculate ratios and width. An embedded computer interfaces with the computer workstation for data transfer. Pulse Width (µs) 29 30

6 Quantification of a Voltage Pulse Data Storage List-Mode Data FSC SSC Event , , Event ,271 30,621 Event ,688 6,189 39, , Data Display Linear Scaling Log Scaling Linear v.s Log Voltage Pulses 0.1 V 0.4 V 1.2 V 4 V 8 V Linear v. Log Amplification Spectral Overlap Compensation Theory 100% Pacific Blue AmCyan PI APC PerCP -Cy7 Linear amplification is usually used for light scatter parameters and DNA analysis. Log amplification is used for fluorescence signals with a large dynamic range. Normalized Intensity 0% Wavelength (nm)

7 Spillover Spillover 530/30 585/42 695/40 -Cy7 780/60 530/30 585/42 695/40 Relative Intensity Relative Intensity Wavelength (nm) -Cy Wavelength (nm) Compensation -% To lower cluster, increase value. Compensation Examples Incorrect Compensation Correct Compensation Undercompensation Overcompensation 530/30 585/42 695/40 -% Relative Intensity Wavelength (nm) Review Data Processing SSC FSC Application Examples APC 41

8 Applications Phenotype Analysis Phenotype Analysis (Cell Surface Antigens/Markers) Intracellular Analysis -- Eg. Cytokines, Signal Transduction molecules etc. DNA Analysis -- Eg. Viability, Cell cycle, Apoptosis etc. Cell Fuction Analysis -- Eg. Free radicals, Ca 2+, Reporter genes etc. CBA (Cytometric Bead Array) Ligand Receptor Adhesion molecule etc Lymphocyte Immunophenotyping Peripheral White Blood Cells CD45 + Monocytes Granulocytes Monocytes T Helper T Lymphocytes B CD3 + CD3 - CD19 + T Cytotoxic CD3 + CD4 + CD3 + CD8 + NK CD3 - CD16 + CD56 + Neutrophils Basophils Eosinophils Intracellular Analysis Permeabilizing solution Cytokine Enzyme signal transduction molecule etc. Cytokine Detection Fixation solution 47 Picture From 48

9 LPS T cell Ionomycin PHA Con A Combination of Cell Surface and Cytoplasmic Staining Th1/Th2/Th17 Phenotyping Kit CD28 CD3 Stimulation Intracellular Staining To enhance the accumulation of intracellular cytokines. Secretion stop Monensin: Cytokines accumulate in the ER Brefeldin A: in Golgi complex. (Brefeldin A or Monensin) Only in vitro To maintain structural integrity. Formaldehyde or glutaraldehyde Keep the protein structure and doesn't change the (accessibility of the) epitopes too much Permeabilisation Saponin (permeablisation buffer). Fixation Signal Transduction Intracellular Staining in Activated Lysed Whole Blood DNA Analysis Cell Cycle Analysis Ethanol Detergent G 2 M G 0 s G 1 G 0 G 1 Nucleic Acid Dye C o u n t s G 2 M N 4N DNA content 53 54

10 Apoptosis (Sub G1) Cell Function Analysis Membrane Potential (DiOC6, JC-1) Oxidative Metabolism (Free Radicals) Intracellular PH Value (Snarf-1) Ca++ Influx (Fluo-4/Fura Red, Indo-1) Phagocytosis Cell Proliferation (PI, BrdU, Intracellular Cyclins) Apoptosis (Annexin V, active Caspase-3) Annexin V Assay Annexin V/PI Double Staining Bordón et al. Radiation Oncology : Cytometric Beads Array (CBA) Beads Provide a Flexible Platform Single Step Incubation or Two-Step Incubation Multiple sizes Different fluorescence intensities Different colors with different intensities 59 60

11 Advantages of Bead-Based Immunoassays Analyze multiple analytes simultaneously Reduced sample volume requirements Reduced hands-on time by parallel analysis of samples Wide dynamic range of fluorescence detection (requires fewer sample dilutions) Proteins Measured A. Interleukin (IL)-2 B. IL-4 C. IL-5 D. IL-10 E. Tumor Necrosis Factor- F. Interferon Cytometry Beads Array (CBA) Standard Curves CBA Flex Sets Open configuration (Up to 30 plex) Clustering based on Red and NIR fluorescence intensity Need to be used at dual-laser(488nm blue v.s 633nm red) instrument CBA Functional Beads Can be conjugated with any Ab or Ag 65 66

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