Flourescence Activated Cell Sorting

Definitions Flow Cytometry Measuring properties of cells in flow Flow Sorting Sorting (separating) cells based on properties measured in flow Also called Fluorescence-Activated Cell Sorting (FACS)

Existing Cell Sorting methods Sedimentation Density gradient sedimentation Affinity extraction Magnetic beads using antibodies FACS

When to use FACS? When very high purity (95%-100%) of the target population is required. For separations on the basis of internal cell staining e.g. of DNA, or of internal antigens, or fluorescent proteins. For enrichment of populations on the basis of surface receptor density. For separation of populations that have a low density of receptors on their surface. When single cell sorting is required (cloning). When other separation methods fail. Bulk separation methods should be used when the starting cell number is greater than ~300 million cells (> 3-10h).

History The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner,Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory

Principle of FACS Target cells as single cell suspension are stained by fluorescent dyes. Laser interrogation and signal processing followed by sort decision Hydrodynamic focusing in a nozzle vibrated by a transducer produces a stream breaking into droplets. Electronic delay until cell reaches break off point. Then the stream is charged + or – Charged droplets deflect by electrostatic field from plates held at high voltage (+/- 5000 volts). http://www.iupui.edu/~wellsctr/MMIA/flow_cytometry/flowcytometry_revised.swf

Cellular parameters measured by FACS Intrinsic Extrinsic No reagents or probes required (Structural) Cell size (Forward Light Scatter) Cytoplasmic granularity (90o Light Scatter) Photosynthetic pigments Reagents are required. Structural DNA content DNA base ratios RNA content Functional Surface and intracellular receptors. DNA synthesis DNA degradation (apoptosis) Cytoplasmic Ca++ Gene expression

Flourochromes Nucleic acid dyes Dyes conjugated antibodies Dyes conjugated proteins Dyes conjugated enzyme substrates Indicator dyes for ions Ca+2, Na+, K+ Dyes for cytoskeletal proteins Dyes for functional organelles

Fluorochromes based on lasers Blue argon laser (488 nm) This is an air cooled laser and therefore cheaper to set up and run. It is the most commonly available laser on single laser machines. Green (usually labelled FL1): FITC, Alexa Fluor 488, GFP, CFSE, CFDA-SE, DyLight 488 Orange (usually FL2): PE, PI Red channel (usually FL3): , PerCP-Cy5.5, PE-Alexa Fluor 700, , . Infra-red (usually FL4; not provided by all FACS machines as standard): PE-Alexa Fluor 750, Red diode laser (635 nm) APC APC-Cy7, APC-eFluor 780 Alexa Fluor 700 Cy5 Draq-5 Violet laser (405 nm) Pacific Orange Amine Aqua Pacific Blue DAPI Alexa Fluor 405 eFluor 450 eFluor 605 Nanocrystals eFluor 650 Nanocrystals

Fluorochromes for samples Immunophenotyping with up to 11-color sorting: e.g. Cascade Blue, Amca, FITC, CY3, PE, Cy5, APC, TR, Per-CP, Alexa-dyes, Tandem dyes. Fluorescent protein expression such as: eBFP, eCFP, eGFP, eYFP, Ds-Red. Cell division sorting by BUdR/Hoechst, CFSE or PKH26. Cell-cycle and cell-ploidy sorting: Propidium iodide (PI), Hoechst dyes, DAPI. Stem cells by the Side Population or Rhodamine Dull. Calcium mobilization according Indo-1 fluoerescences. Apoptosis sorting of the sub diploid peak or according Annexin V- staining. Cell volume and morphological characteristics by light scatter parameters and autofluorescence.

Basic Components of Flow Cytometry Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer Fluidics Optics Electronics

Optics Fluidics Electronics

Fluidics: Sheath fluid Target cell concentration (required): ~106 cells/ml The volume of one sorted drop is 1.4 nl (70µm,100kHz, 60psi).106 cells result in 1.4 ml. The flow of FACS begins at Sheath Fluid Reservoir. Sheath fluid is a buffer of composition appropriate for the cells to be used. Eg: phosphate buffer saline solution

Regulation of sample injection Differential pressure system Use gas or air to pressurize sample and sheath Difference in pressure between sample and sheath will control sample volume flow rate Volumetric injection Syringe pump to load sample Sample volume flow rate can be changed by changing speed of motor

Fluidics: Sample injection Sample is injected into a sheath fluid as it passes through a small (50-300 µm) orifice When conditions are right, sample fluid flows in a central core that does not mix with the sheath fluid. This is termed Laminar flow The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing C:\Users\Latha\Desktop\Fluorescence.gif

Fluidics process Single cell suspension, e.g. blood cells or isolated tissue cells. Properties of the target cells were stained by fluorescent dyes. A piezoelectric transducer in the nozzle holder causes the stream with the cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet (4% at 100 kHz and 20.000 cells/s). Just before the stream breaks into droplets the flow passes through the observation point where the fluorescence intensities of each cell are measured by the flow cytometer. At this point the cells for sorting are selected

Optics- Illuminating systems Lasers Provide single wavelength of light. 350-363, 405, 420, 457, 514, 532, 600, 633, 660nm Argon ion, Krypton ion, HeNe, HeCd, YAG, Fluorescent probes absorbs energy from laser and releases absorbed energy by emission of photons at longer wavelength- Stokes Shift Arc Lamps provide mixture of wavelengths that must be filtered to select desired wavelengths Mercury, Mercury-Xenon

Optics- Light Scatter Forward Scatter Side Scatter- 90o

Optics- Light Scatter Forward scatter tends to be more sensitive to surface properties of particles than side scatter can be used to distinguish live from dead cells Cell size α forward scatter Side scatter tends to be more sensitive to inclusions within cells than forward scatter can be used to distinguish granulated cells from non-granulated cells granularity α 90o scatter

Main components of optics Lens, Mirrors, Filters, Detectors Excitation Optics shape and focus laser beam Collection Optics collect and filter wavelengths of light that come from the particle-laser beam interaction The fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel The specificity of detection is controlled by the wavelength selectivity of optical filters and mirrors

Optical Filters Transmit λ> cuton Transmit λ< cut-off Transmit λ around a range

Detectors Two common detector types Photodiode used for strong signals when saturation is a potential problem (e.g., forward scatter detector) Photomultiplier tube (PMT) more sensitive than photodiode but can be destroyed by exposure to too much light

FACS optics 530nm band pass FL1 560nm short pass 585nm band pass 488nm band pass FSC 488nm laser beam 560nm short pass dichroic mirror 585nm band pass FL2 PMT 510nm long pass 488nm band pass SSC PD flow cell

Electronics Processing of signals from detectors Preamplification Strengthen signals so that they can travel from remote detectors to central electronics Amplification Adjust signal intensity Linear or Logarithmic Log transformation can also be performed after digitization using a look-up table

Electronics: Data Acquisition Individual cell fluorescence quanta is picked up by the various detectors(PMT’s). PMT’s convert light into electrical pulses. These electrical signals are amplified and digitized using Analog to Digital Converters (ADC’s). Each event is designated a channel number (based on the fluorescence intensity as originally detected by the PMT’s) on a 1 Parameter Histogram or 2 Parameter Histogram. All events are individually correlated for all the parameters collected.

Common Display formats Histogram single parameter only, array created acquisition and analysis Dot Plot bivariate, two parameters (scattergram) Density Plot bivariate, 64x64, 128x128, or 256x256 2D array Contour Plot analysis only

showing distributions relative numbers of events Display formats Dot Plot acquisition rare events Density Plot showing distributions relative numbers of events Contour Plot showing populations Histograms Bivariate

Example Side Scatter Projection Neutrophils Forward Scatter Monocytes 90 Degree Scatter 200 400 600 800 1000 Lymphocytes Monocytes Neutrophils Side Scatter Projection Forward Scatter

Sorting Enrich Mode: all sorted drops with a positive cell are chosen regardless of contaminats. Purify Mode: contaminating events in the sorted drops will result in an abort decision. Single-Cell Mode: only drops containing one positive cell having a safe zone are sorted.

Measurable parameters volume and morphological complexity of cells cell pigments such as chlorophyll or phycoerythrin DNA (cell cycle analysis, cell kinetics, proliferation, etc.) RNA chromosome analysis and sorting (library construction, chromosome paint) protein expression and localization Protein modifications, phospho-proteins transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent * cell surface antigens (Cluster of differentiation (CD) markers) intracellular antigens (various cytokines, secondary mediators, etc.) nuclear antigens enzymatic activity pH, intracellular ionized calcium, magnesium, membrane potential membrane fluidity apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity) cell viability monitoring electropermeabilization of cells characterising multidrug resistance (MDR) in cancer cells

Will the cells be harmed? Generally, the cells will be not harmed through the process itself as long as they are maintained at a temperature, pH, and in media that is most suited to them. In most cases cells are at least 95% viable after a sort with typical system parameters. What comes out is closely related to what goes in the sorter.

FACS instruments FACSAria II: BD FACSCalibur: It has three excitation lines at 488nm, 633nm, and 407nm. It can collect up to nine fluorescent parameters. It can sort 1-4 separate population simultaneously or perform single cell sorting into 96 well plates. Sorts 90000 – 30000cells/sec BD FACSCalibur: It has two excitation lines at 488nm and 633nm. It can collect up to four fluorescent parameters. Recent: MoFlo XDP Cell Sorter from Beckman Coulter