1. Flourescence Activated Cell Sorting

2. 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)

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

4. 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).

5. 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

6. 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 (+/ volts).

7. 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

8. 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

9. 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

10. 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.

11. 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

12. Optics
Fluidics
Electronics

13. 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

14. 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

15. Fluidics: Sample injection
Sample is injected into a sheath fluid as it passes through a small ( 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

16. 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 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

17. Optics- Illuminating systems
Lasers
Provide single wavelength of light.
, 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

18. Optics- Light Scatter
Forward Scatter
Side Scatter- 90o

19. 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

20. 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

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

22. 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

23. 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

24. 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

25. 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.

26. 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

27. 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

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

29. 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.

30. 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

31. 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.

32. 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 – 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