flourescence activated cell sorting. definitions flow cytometry measuring properties of cells in...
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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
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
Intrinsic Extrinsic
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 lasersBlue 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
FluidicsFluidics
OpticsOptics
ElectronicsElectronics
FluidicsFluidics
OpticsOptics
ElectronicsElectronics
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
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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
488nm band pass FSC488nm laser beam
560nm short passdichroic mirror585nm band pass
FL2PMT
510nm long passdichroic mirror
488nm band passSSC
PMT
PMT
PDflow 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)
acquisition and analysis
Density Plot
bivariate, 64x64, 128x128, or 256x256 2D array
acquisition and analysis
Contour Plot
bivariate, 64x64, 128x128, or 256x256 2D array
analysis only
Display formats
Dot Plot
acquisition
rare events
Density Plot
showing distributions
relative numbers of events
Contour Plot
showing populations
relative numbers of events
Histograms Bivariate
Example
90 Degree Scatter0 200 400 600 800 1000
0 2
00 4
00 6
00 8
0010
00
Lymphocytes
Monocytes
Neutrophils
Side Scatter Projection
For
war
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catte
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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:
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