Transcript Flourescence Activated Cell Sorting

Flourescence Activated Cell
Sorting
Definitions
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Flow Cytometry
 Measuring properties of cells in flow
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Flow Sorting
 Sorting (separating) cells based on properties
measured in flow
 Also called Fluorescence-Activated Cell Sorting
(FACS)
Existing Cell Sorting methods
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Sedimentation
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Density gradient sedimentation
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Affinity extraction
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Magnetic beads using antibodies
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FACS
When to use FACS?
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When very high purity (95%-100%) of the target population is required.
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For separations on the basis of internal cell staining e.g. of DNA, or of internal antigens, or
fluorescent proteins.
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For enrichment of populations on the basis of surface receptor density.
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For separation of populations that have a low density of receptors on their surface.
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When single cell sorting is required (cloning).
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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
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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
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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.
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Laser interrogation and signal processing
followed by sort decision
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Hydrodynamic focusing in a nozzle vibrated by a
transducer produces a stream breaking into
droplets.
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Electronic delay until cell reaches break off
point. Then the stream is charged + or –
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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
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No reagents or probes required
(Structural)
Extrinsic
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Reagents are required.
 Structural
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Cell size (Forward Light
Scatter)
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Cytoplasmic granularity (90o
Light Scatter)
Functional
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Photosynthetic pigments
DNA content
DNA base ratios
RNA content
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Surface and intracellular
receptors.
DNA synthesis
DNA degradation
(apoptosis)
Cytoplasmic Ca++
Gene expression
Flourochromes
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Nucleic acid dyes
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Dyes conjugated antibodies
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Dyes conjugated proteins
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Dyes conjugated enzyme substrates
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Indicator dyes for ions
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Ca+2, Na+, K+
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Dyes for cytoskeletal proteins
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Dyes for functional organelles
Fluorochromes based on lasers
Blue argon laser (488 nm)
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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)
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APC
APC-Cy7, APC-eFluor 780
Alexa Fluor 700
Cy5
Draq-5
Violet laser (405 nm)
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Pacific Orange
Amine Aqua
Pacific Blue
DAPI
Alexa Fluor 405
eFluor 450
eFluor 605 Nanocrystals
eFluor 650 Nanocrystals
Fluorochromes for samples
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Immunophenotyping with up to 11-color sorting: e.g. Cascade Blue, Amca, FITC,
CY3, PE, Cy5, APC, TR, Per-CP, Alexa-dyes, Tandem dyes.
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Fluorescent protein expression such as: eBFP, eCFP, eGFP, eYFP, Ds-Red.
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Cell division sorting by BUdR/Hoechst, CFSE or PKH26.
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Cell-cycle and cell-ploidy sorting: Propidium iodide (PI), Hoechst dyes, DAPI.
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Stem cells by the Side Population or Rhodamine Dull.
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Calcium mobilization according Indo-1 fluoerescences.
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Apoptosis sorting of the sub diploid peak or according Annexin V- staining.
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Cell volume and morphological characteristics by light scatter parameters and
autofluorescence.
Basic Components of Flow
Cytometry
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Cells in suspension
flow in single-file through
an illuminated volume where they
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scatter light and emit fluorescence
that is collected, filtered and
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converted to digital values
that are stored on a computer
Fluidics
Optics
Electronics
Optics
Fluidics
Electronics
Fluidics: Sheath fluid
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Target cell concentration (required): ~106 cells/ml
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The volume of one sorted drop is 1.4 nl (70µm,100kHz, 60psi).106 cells result in 1.4
ml.
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The flow of FACS begins at Sheath Fluid Reservoir.
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Sheath fluid is a buffer of composition appropriate for the cells to be used.
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Eg: phosphate buffer saline solution
Regulation of sample injection
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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
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Volumetric injection
 Syringe pump to load sample
 Sample volume flow rate can
be changed by changing speed
of motor
Fluidics: Sample injection
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Sample is injected into a sheath fluid as it passes
through a small (50-300 µm) orifice
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When conditions are right, sample fluid flows in a
central core that does not mix with the sheath
fluid. This is termed Laminar flow
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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
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Single cell suspension, e.g. blood cells or isolated tissue cells.
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Properties of the target cells were stained by fluorescent dyes.
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A piezoelectric transducer in the nozzle holder causes the stream with the
cells to break into individual droplets.
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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).
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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
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Lasers
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Provide single wavelength of light.
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350-363, 405, 420, 457, 514, 532, 600, 633, 660nm
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Argon ion, Krypton ion, HeNe, HeCd, YAG,
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Fluorescent probes absorbs energy from laser and releases absorbed energy by emission of photons at
longer wavelength- Stokes Shift
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Arc Lamps
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provide mixture of wavelengths that must be filtered to select desired wavelengths
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Mercury, Mercury-Xenon
Optics- Light Scatter
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Forward Scatter
Side Scatter- 90o
Optics- Light Scatter
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Forward scatter tends to be more sensitive to surface properties of particles
than side scatter
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can be used to distinguish live from dead cells
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Cell size α forward scatter
Side scatter tends to be more sensitive to inclusions within cells than forward
scatter
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can be used to distinguish granulated cells from non-granulated cells
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granularity α 90o scatter
Main components of optics
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Lens, Mirrors, Filters, Detectors
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Excitation Optics
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shape and focus laser beam
Collection Optics
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collect and filter wavelengths of light that come from the particlelaser beam interaction
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The fluorescence emitted by each fluorochrome is usually detected
in a unique fluorescence channel
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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
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Two common detector types
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Photodiode
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used for strong signals when saturation is a potential
problem (e.g., forward scatter detector)
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Photomultiplier tube (PMT)
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more sensitive than photodiode but can be destroyed by
exposure to too much light
FACS optics
PMT
530nm band pass
FL1
585nm band pass
FL2
PMT
560nm short pass
dichroic mirror
488nm band pass
SSC
PMT
510nm long pass
dichroic mirror
488nm band pass FSC
488nm laser beam
flow cell
PD
Electronics
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Processing of signals from detectors
 Preamplification
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Strengthen signals so that they can travel from remote
detectors to central electronics
Amplification
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Adjust signal intensity
Linear or Logarithmic
Log transformation can also be performed after
digitization using a look-up table
Electronics: Data Acquisition
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Individual cell fluorescence quanta is picked up by the various
detectors(PMT’s).
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PMT’s convert light into electrical pulses.
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These electrical signals are amplified and digitized using Analog to
Digital Converters (ADC’s).
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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.
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All events are individually correlated for all the parameters collected.
Common Display formats
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Histogram
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single parameter only, array created
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acquisition and analysis
Dot Plot
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bivariate, two parameters (scattergram)
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acquisition and analysis
Density Plot
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bivariate, 64x64, 128x128, or 256x256 2D array
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acquisition and analysis
Contour Plot
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bivariate, 64x64, 128x128, or 256x256 2D array
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analysis only
Display formats
Dot Plot
Density Plot
Contour Plot
acquisition
showing distributions
showing populations
rare events
relative numbers of events
relative numbers of events
Histograms
Bivariate
Example
Scatter
Forward
1000
800
600
Side Scatter Projection
Neutrophils
400
Monocytes
0
200
Lymphocytes
0
200
400
600
800
90 Degree Scatter
1000
Sorting
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Enrich Mode: all sorted drops with a positive cell are chosen
regardless of contaminats.
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Purify Mode: contaminating events in the sorted drops will result in
an abort decision.
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Single-Cell Mode: only drops containing one positive cell having a
safe zone are sorted.
Measurable parameters
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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?
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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
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FACSAria II:
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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:
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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