Flow Cytometry -Introduction and applications Flashcards
What is flow cytometry?
A technique which allows us to simultaneously measure several physical characteristics to a single cell in suspension.
Done by light scatter and fluorescence
What is the definition of flow cytometry?
Measuring properties of cells in flow
What is the definition of flow sorting ?
Sorting (separating ) cells based on properties measured in flow.
This is also called Fluorescence-Activated Cell Sorting (FACS)
What information can flow cytometer tell us about a cell?
- Relative size
- Relative Granularity/Internal Complexity
- Relative fluorescence intensity (using markers)
What markers can be measured ?
- Cell surface receptors
- Intercellular cytokines
- DNA-Apoptosis/cell cycle/viability
What are two methods of visualisation?
- Flow cytometry
- Fluorescence Microscopy
What technique can we use to quantitate cells?
-We can use flow cytometry which can also quantitate the fluorescence
How does a flow cytometry work?
Fluidics :
-Cell in suspension flow in a single file through.
Optics:
-An illuminated volume where they scatter light ,emit fluorescence that is collected and filtered.
Electronics:
This is converted into digital values that are stored on a computer
(Then analysed )
Outline the different processes involved in flow cytometry
Light source Flow chamber Optical system Light detectors Computer
Describe the Fluidics phase
- The cells need to be in suspension flow in single file.
- The sample is injected into sheath fluid as it passes through small orifice (opening)
- Sample fluid flows in a central core that does not mix with the sheath fluid (Laminar flow)
-Introduction of a large volume into a small volume
(Hydrodynamic Focusing)
Describe the Light sources for the Optic stage.
Light sources are lasers:
- Single wave length of light (a laser line ) or mix of wavelengths
- Can provide light (milliwatts -watts)
- Inexpensive air-cooled units or expensive water cooled units
- emit coherent light (single frequency)
Describe light scatter
This happens when light hits the cell.
-Most common wavelength of laser is 488nm.
- The light becomes scattered in a forward direction (FSC) and this is proportional to cell size
- 90 degrees light scatter proportional to granularity (SSC) Side scatter
Describe dot plot
X axis is forward scatter (size)
Y axis side scatter (granularity )
Each dot is a cell.
We can identify different type of cells based on their size and granularity.
The scatter shows this information
What is Granularity ?
This is a cells internal complexity
Describe the passage of Laser-based flow Cytometry
1.Flow cell - cells leaving the nozzle tip are hit by the laser
2.The cells have been labelled using fluorochromes
They emit light in different colours .
3.There is an overlap in emission spectrum of each fluorochrome.
3.Complex system of mirrors and filters is used to restrict the amount of light hitting the PMT (photomultiplier tube ) = we can differentiate the fluorescence from each fluorochrome separately
What is the electronic stage of flow-cytometry?
- This is the final stage
- The PMT will convert light signals into digital signals.
- Analogue- digital conversion
What are the key differences between flow cytometry and fluorescence microscopy?
- Fluorescence microscopy (FM) has fields which means its not quantitative as you would need to look at many fields to quantitate cells accurately.
- Flow cytometer(FC) can analyse 1000s every second
Rare cells can be quantitated accurately in FC
Brightness of cells is difficult by eye using FM but the machine in FC will do it and quantitate the amount of fluorescence each cell
How does fluorescence happen?
What is this process called?
A laser hits a fluorochrome ,exciting it at a wavelength .It then goes back to to its unexcited state and emits fluorescence at a higher wavelength.
This is called excitation and emission spectrum
What is stokes shift ?
The energy difference between the lowest energy peak of absorbance and the highest energy of emission
What are some different fluorochromes and dyes ?
FITC (Fluorescein isothiocyanate)- Green
PE (Phycoerythrin)-Orange
PerCP (Peridinin Chlorophyll Protein)-Red
What are the wavelengths which each of these fluorochromes emit /excited ?
FITC - Excited at 488 and emits 520
PE-Excited at 488 and emits 580
PerCP- Excited at 488 and emits at 620
What is fluorescence ?
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation
Why can different fluorochromes be used together and what is a benefit of this ?
We can use three all excited by the same laser but they emit at different wavelengths.
The emission always overlaps therefore the filters and mirrors will filter out the overlapping light so we can analyse data from all fluorochromes together
What are some cells which are in single cell suspension ?
Peripheral blood Bone Marrow Fine Needle aspirate CSF and other fluids Fresh tissue
What are the two ways to label cells with monoclonal antibodies /fluorocromes?
Direct :Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes.
Indirect: Unconjugated MoABs
How is the data displayed when converted from light to digital ?
1.Histogram -One dimensional display (cell number=y axis /fluorescence intensity =x axis (one parameter)
2.Dot plot -Two dimensional
side scatter =x axis forward scatter=y axis
(Two parameters)- We could potentially identify four populations
What are two things could do to the data we have collected ?
Gating
Analysis
What is Gating ?
-Two dimensional dot plot
Draw a gate around the population we want to look at further.
Then we will only display the gated cells
What is Analysis?
- Analysis
- We will only measure cells satisfying the gate -only analysing one parameter.
Using a marker we can see what proportion of the cells that are negative/positive for it .
Outline how increasing fluorochromes can increase identified populations
1 fluorochrome =1 population
2 fluorochrome= 4 populations
3 fluorochrome =-8 populations
Cells which are positive for all or negative for all
How can we analyse cell cycles?
Cellular DNA is detected using a fluorescent dye which binds to DNA
Propidium iodide is used -increases fluorescence when binds to DNA
Requires permeabilization of the plasma membrane so the PI can enter
How is propidium iodide used ?
It is excited by a 488 laser and emits at 620nm
What are the different stages of the cell cycle?
G0, G1,S,G2,M
What is propidium iodide fluorescence proportional to ?
The amount of DNA in a cell
The G2M phase will have the highest fluorescence and will have double the DNA
How can we measure cell viability ?
- PI can not cross a cell membrane
- If the PI penetrates the cell membrane the cell is assumed damaged
- Cells that are brightly fluorescent with PI are damaged or dead cells
Describe apoptosis briefly
Apoptosis is a programmed cell death where the cell goes through regulated process of dying
Characteristics are condensation of the chromatin material
Blebbing of nuclear material
Accompanied by internucleosomal degradation of DNA which gives rise to ladder pattern on DNA gel electrophoresis
How can we detect apoptosis ?
What are the three methods ?
By staining using the dye PI -cells are fixed
Phosphatidyl serine (this is usually inside but during apoptosis will be on the outside ) can be detected by incubating the cells with fluorescien-labeled Annexin V,PI
By staining with 7-aminoactinomycin(cell not fixed)
Describe the PI dye method of detecting apoptosis
The Sub -G0 peak
This is a peak which is found in apoptotic cells by staining using PI.
Issue is not all cells will have this peak and
It might all be DNA fragments
Describe the Annexin V + PI method of detecting apoptosis
The Phosphatidyl serine normally sits inside the cell.
During early apoptosis :
-The PS will be outside meaning the Annexin V can bind to it however the cell membrane is in tact
The cell is positive for Annexin V but negative for PI
During late apoptosis :
The cell will be positive for both Annexin V and PI as cell membrane is no longer in tact
Describe the 7-Aminoactinomycin D method -7-AAD (apoptosis )
- Excited at 488nm
- Emitted at 660nm
DNA specific
Intercalates in G-C regions
Long emission wavelength
A single dye -allows us to use two other fluorochromes to evaluate cell surface antigens as well as apoptosis
List some applications of flow cytometry.
- Immunophenotyping of leukaemias & lymphomas
- Detection of MRD
- Stem cell enumeration
CD4/CD8 in HIV - Measurement of intracellular cytokines
- Study of cell cycle, viability & apoptosis
- Measurement of cell proliferation
- Assessment of transfection efficiency
Describe the fine-tuning of cell sorting
The basics of flow cytometry are present: the cells are in a suspension in a single file, a laser is put on them, causing them to emit light that is a picked up and tells a computer connected to it what type of cell it is.
In cell sorting, we can draw a range around the types of cells we want, so the computer will ‘sort them out’.
The nozzle tip is always vibrating, so much that the stream breaks off into droplets at some point. Within milliseconds of detection of a certain cell, if it is the type that we want, the computer will charge that cell when it is at the end of a droplet, breaking it off.
The cell is then collected into a tube by being pulled towards a deflection plate (due to its charge). The rest of the cells the go to be collected for waste.
As a result, we get a sample of very purified cells, with endless possibilities in research.
Describe the simplest univariate cell cycle method.
In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane.
