FLOW CYTOMETRY AND CELL SORTING Technical Seminar FLOW CYTOMETRY AND CELL SORTING Bill Hyun Jane Gordon Sarah Elmes

Laboratory for Cell Analysis (LCA) Over 450 Users of LCA shared instrumentation - More than 40 current clinical and research projects - LCA provides support to over $10 Million of annual UCSF extramural funding Cytometry support for Bay Area hospitals, biotech companies, research foundations, and other academic institutions Northern California Cytometry Group LCA Cytometry Courses – Image and Flow LCA Protocol Library

LCA/LBNL Flow Cytometers FACScan 3 - color, 5 - parameter benchtop analyzer FACS Calibur (three units) 4 - color, dual laser (488 and 633nm) Sample loader - walk away FACS Vantage SE DIVA 9-parameter, 3-laser optical bench High speed sorting - rare event Auto-cloning - 96 well plate Index sorting and abort-save

Technical Seminar Principles of Flow Cytometry - Features - Flow Chamber - Lasers and Optics - Fluorescence and Light Scatter - Electronics - Data Processing Data Acquisition and Handling - Philosophy - Data Analysis Cell Sorting

What is Flow Cyto/metry?? Flow cytometry is a technology that allows the SIMULTANEOUS MEASUREMENT OF MULTIPLE PHYSICAL CHARACTERISTICS OF A SINGLE CELL. These measurements are made on a per cell basis at routine rates in a moving stream. Mack Fulwyler, 1982

Flow Cytometry – Partial History 1934 Moldavan – red blood cells measured in a microscope with a capillary flow and a photodetector 1945 Reynolds – laminar flow system 1947 Coulter – patent for cell counter 1964 Kamentsky – 500 cells/second measured for light scatter and uv absorption 1965 Fulwyler – electrostatic cell sorter based on volume 1966 Van Dilla – sorting based on DNA content 1969 Hulett et al – sorting based on cell fluorescence 1972 BDIS – first commercial cell sorter 1975 Gray – chromosome sorting

What can flow cytometry do? Enumerate particles in suspension Evaluate 105 to 106 particles/cells in less than 1 min Measure particle-scatter as well as innate fluorescent Measure 2o fluorescence from multiple fluorochromes Sort single particles/cells for subsequent analysis or growth Separate “live” from “dead” particles Give operators white hair

Fluorescent Cell Properties

Optical Design Laser Sample Dichroic Filters Flow cell Bandpass PMT 5 PMT 4 Sample PMT 3 Dichroic Flow cell Filters PMT 2 Scatter PMT 1 Sensor Bandpass Laser Filters

Scatter Pattern of Human leukocytes A flow cytometry scattergram Lymphocytes Monocytes Neutrophils Forward scatter (size) Side scatter (granularity)

Flow cytometry measurements SCATTER FLUORESCENCE IMAGE G M L

Instrument Components Fluidics: To introduce and restrict the cells for interogation. Controls specimen, sorting, rate of data collection Optics: An excitation source and emission optics to generate and collect light signals. Light source(s), detectors, spectral separation Electronics: To convert optical signals to proportionate electronic signals and digitize them for computer analysis. Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

Fluorescence Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission Stokes Shift is 25 nm Fluorescein molecule 495 nm 520 nm Fluorescnece Intensity Wavelength

Common Fluorescent Probes 600 nm 300 nm 500 nm 700 nm 400 nm 457 350 514 610 632 488 PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid

Fluorescence signal separation

Spectral Overlap

Compensation FITC Beads Alone No Compensation Three Beads Electronically compensated display

Resonance Energy Transfer Fluorescence Resonance Energy Transfer Molecule 1 Molecule 2 Fluorescence Fluorescence ACCEPTOR DONOR Intensity Absorbance Absorbance Wavelength

Fluidics

Cell Sorting SORT DECISIONS -4KV Frequency Histogram +4KV LEFT RIGHT Sample in Sheath Stream Charge The central component of a flow cytometer is the flow cell. A cutdown of a typical flow cell indicates the salient features. Sample is introduced via the sample insertion rod. Sheath fluid (usually water or saline) is introduced to surround the insertion rod causing hydrodynamic focusing of flowing cells which are contained within a core fluid. The laser intersects the fluid either outside the flowcell (in air) or in a slightly extruded portion of the flow cell tip (in quartz). Piezoelectric crystal oscillator Sheath in Sensors SORT DECISIONS Laser beam SMALL BEAD LARGE BEAD Sensor SORT LEFT SORT RIGHT Last attached droplet +4KV -4KV Frequency Histogram LEFT RIGHT Signals are collected from several sensors placed forward or at 90° to the laser beam. It is possible to “sort” individual particles. The flow cell is resonated at a frequency of approximately 32KHZ by the piezoelectric crystal mounted on the flow cell. This causes the flowing stream to break up into individual droplets. Gating characteristics can be determined from histograms (shown right) and these can be used to define the sort criteria. These decisions are all controlled by the computer system and can be made at rates of several thousand per second. SMALL BEAD Waste LARGE BEAD

SORTING

Data Presentation Formats Histogram Dot plot Contour plot 3D plots Dot plot with projection Overviews (multiple histograms)

Data Analysis Concepts Gating Single parameter Dual parameter Multiple parameter Back Gating Note: these terms are introduced here, but will be discussed in more detail during analysis

Data Representation Tube ID FITC Fluorescence CD45 CD8 CD4 Mo1 leu11a

Chromosome ID and Sorting

The Cell Cycle M G2 G1 G0 Quiescent cells S

A DNA histogram G0-G1 Cell Number G2-M S Fluorescence Intensity 14

A typical DNA Histogram G0-G1 G2-M S # of Events Fluorescence Intensity 17

Reticulocyte Analysis REtics Reticulocyte Analysis RMI = 34 RMI = 0 use of maturity index decreases interlab variability RMI = fraction of highly fluorescent retics/total retics cursor is set to exclude nucleated cells .1 1 10 100 1000 .1 1 10 100 1000 log Thiazole Orange log Thiazole Orange

Labeling Strand Breaks with dUTP Green: apoptotic cells R2: Apoptotic Cells Green Fluorescence Side Scatter R1: Normal Cells Red: normal cells PI-Red Fluorescence [Fluorescein-deoxyuridine triphosphate (dUTP)] Forward Scatter Green Fluorescence Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidin Red fluorescence is DNA counter-stained with 20µg/ml PI

Three Color Lymphocyte Patterns CD4 CD4 CD3 CD8 When more than two antibodies are combined, additional subsetting of the cells can be achieved. If one antibody is used to identify a specific lineage of cells, i.e. CD3, five subsets of T-cells are easily resolved, when CD4 vs C8 is displayed. CD8 CD3 Data from Dr. Carleton Stewart

Run on Coulter XL cytometer mixture Run on Coulter XL cytometer mixture Scatter BG BG E.coli Scatter E.coli Yo-Yo 1 - 488 ex membrane impermeant so cells must be fixed. Fix in ethOH - stain 5 minutes run flow Spore coat difficult to get dyes in. Spores have less fluorescent than vegetative bacteria Fluorescence YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores.

Live cell/dead cell PI Fluorescence Hoechst 33342 PI Data from Dr. Doug Redelman, Sierra Cytometry

Superoxide Hydroethidine Hydrogen Peroxide Dichlorofluorescein Oxidative Reactions Superoxide Hydroethidine Hydrogen Peroxide Dichlorofluorescein Glutathione levels Monobromobimane Nitric Oxide Dichlorofluorescein

Calcium Flux Flow Cytometry Image Cytometry Time (seconds) Stimulation 0.8 0.7 0.6 0.5 Ratio: intensity of 460nm / 405nm signals 0.4 0.3 0.2 Stimulation 0.1 36 72 108 144 180 Time (seconds) Time (Seconds) 50 100 150 200

Membrane Potential Oxonol Probes Cyanine Probes How the assay works: Carbocyanine dyes released into the surrounding media as cells depolarize Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs Time (sec) Green Fluorescence Repolarized Cells 0 512 1024 300 150 0 1200 2400 PMA Added fMLP Added Depolarized Cells

Sorting and cell isolation Summary Main Applications DNA and RNA analysis Phenotyping Cell Function Sorting and cell isolation Immunological assays