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

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

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

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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