Flow Cytometry Flashcards
Explain the basic principles of flow cytometry
Flow cytometry is a technology that allows rapid analysis of multiple physical and chemical characteristics of single cells or particles as they flow in a fluid stream through a beam of light
1) Suspension of Cells or Particles:
- For flow cytometry to work, the cells or particles of interest must be in a liquid suspension
- This can be a simple saline solution or a complex buffer designed to protect the cells
2) Interrogation Point or Flow Cell:
- The suspension of cells is then passed, usually in single file, through an interrogation point, often a flow cell, where they are hit with a beam of light, typically from a laser
3) Scatter:
- When the cells pass through the light beam, they scatter light in all directions
- The pattern and quantity of scattered light can tell us the size and structure of the cells
- Light scattered in the same direction as the laser beam (forward scatter or FSC) is proportional to the cell size
4) Fluorescence:
- In addition to scatter, cells or particles that have been stained with fluorescent dyes or antibodies will also emit light at a longer wavelength than the source light
- This can be measured to determine the presence and quantity of various cellular components or markers
5) Detectors:
- The scattered and emitted light is captured by detectors
- Forward scatter and side scatter detectors capture scatter data, and additional detectors capture the fluorescence signals
- Each detector only captures light at specific wavelengths, allowing for the detection of different fluorophores
6) Data Analysis:
- The information from the detectors is processed by a computer to generate the output data
- Each cell is represented as a dot in a plot, with its position determined by the intensity of light for the parameters being measured
Describe the major components of flow cytometry instrumentation
1) Fluidics System:
- This system is responsible for transporting cells in a stream to the laser beam for analysis
- typically composed of a sample injection chamber where cells are introduced,
- a sheath fluid reservoir containing a saline or buffered solution that carries cells single-file to the interrogation point
- and pumps or pressure sources to control fluid flow
- Hydrodynamic focusing, where sheath fluid surrounds the sample stream and forces it to become narrower, allows cells to pass through the laser beam one at a time
2) Optics System:
- This includes the light source, usually one or more lasers, that illuminates the cells or particles as they flow past
- The optics system also includes a collection of lenses and filters that direct and focus the light source onto the sample, and then collect and correctly filter the resulting signals (both scattered and fluorescent light)
- Forward Scatter Detector: Light scattered in the direction of the laser path (forward scatter) is collected and is usually used as a measure of particle size
- Side Scatter Detector: Light scattered perpendicular to the laser path (side scatter) gives information on the cell’s granularity or complexity
- Fluorescence Detectors: These detectors collect emitted light after excitation by the laser. The light is separated by wavelength using a combination of dichroic mirrors and bandpass filters, allowing specific fluorescence signals to be detected and measured
**3) Electronics system:
- The electronic signals (photons of light converted to electronic signals by photodetectors, such as photomultiplier tubes or avalanche photodiodes) are processed in this part of the system
- This includes amplification of the signals, conversion of the analog signals to digital format, and then routing these signals to a computer for analysis
- The data are often displayed as histograms or dot plots
4) Data Analysis Software
- This component allows the user to view and analyse the data collected by the flow cytometer
Describe the principles of fluorescence and some commonly used fluorochromes
Fluorescence is a phenomenon by which a molecule absorbs light energy (photons) of a particular wavelength and then emits light at a longer wavelength
I.e. The molecule, upon absorption of a photon, jumps to an excited state and then returns to its ground state by emitting the extra energy as a photon of light. The emitted light is what we measure as fluorescence
In flow cytometry and fluorescence microscopy, this principle relies on the use of fluorescent molecules or fluorochromes, which are capable of emitting light upon light excitation
Fluorescein isothiocyanate (FITC): It absorbs light at a wavelength of 495 nm and emits light at a wavelength of 519 nm (green)
Phycoerythrin (PE): PE is a protein fluorochrome that absorbs light at a wavelength of 496 nm, 546 nm and emits light at a wavelength of 576 nm (orange/red)
Peridinin-chorophyll-protein complex (PerCP): multiple absorbance maxima and emits light at a wavelength of 675 nm (far red)
Allophycocyanin (APC): absorbs light at a wavelength of 650 nm, 660 nm and emits light at a wavelength of 660 nm (red)
Discuss staining of surface and cytoplasmic antigens
Staining of surface and cytoplasmic antigens is often carried out for flow cytometry or immunofluorescence microscopy.
Each of these types of antigen resides in a different part of the cell and therefore require different protocols for successful staining
1) Surface Antigen Staining:
- This is relatively straightforward because the antigens are readily accessible to the antibodies used for staining
- Cells are typically suspended in a buffer solution, and fluorescently labelled antibodies that recognise the surface antigens of interest are added
- The antibody and cells are incubated together to allow the antibody to bind to the antigen
- After incubation, the cells are washed to remove unbound antibodies, and then they are ready for analysis
- For flow cytometry, surface antigens are typically stained in their native conformation on live cells, as fixation can sometimes alter the antigen and prevent the antibody from binding
2) Cytoplasmic (and Nuclear) Antigen Staining:
- Staining these antigens are more complex because the cell membrane needs to be permeabilised to allow the antibodies to access the intracellular antigens
- After the cells have been fixed (typically with a cross-linking agent like formaldehyde) to preserve cellular structures, a permeabilising agent (like saponin or Triton X-100) is added to the cells to poke holes in the membrane
- The fluorescently labelled antibodies can then be added to the cells and allowed to bind to their antigens
- After incubation, the cells are washed to remove unbound antibodies, and then they are ready for analysis
Explain DNA staining
involves the use of substances called DNA stains or DNA dyes, which bind to DNA and can then be visualized under certain types of light
These dyes often fluoresce when exposed to UV or specific wavelengths of light, thus allowing the detection and visualization of the DNA under a microscope
1) Intercalating Stains:
- These are planar molecules that can insert themselves between the base pairs of the DNA helix, which distorts the DNA structure and often results in an enhanced fluorescence signal
- Examples include ethidium bromide (EtBr), propidium iodide (PI), and DAPI
- They bind to the minor groove of the DNA double helix
2) Minor Groove Binding Stains:
- These dyes bind to the minor groove of DNA but do not cause as much distortion as intercalators
- These dyes bind to the minor groove of DNA but do not cause as much distortion as intercalators
3) Miscellaneous Stains:
- These include dyes like acridine orange, which can bind to both DNA and RNA but fluoresce at different wavelengths depending on which nucleic acid they are bound to
Discuss clinical and research applications of flow cytometry
1) Immunophenotyping:
- used to study the protein expression in cells, typically on the cell surface, to identify cell types, subsets and activation states
- In clinical diagnostics, this is commonly used in hematology to diagnose, classify and monitor diseases such as leukemia and lymphoma, based on the presence or absence of specific markers on the surface of cells
2) Cell Cycle Analysis:
- By staining DNA with a fluorescent dye, researchers can use flow cytometry to analyze the cell cycle
- This allows the identification of cells in different phases of the cell cycle (G0/G1, S, and G2/M)
3) Apoptosis and Necrosis Detection:
- Using specific markers that change when a cell undergoes apoptosis (programmed cell death) or necrosis (accidental cell death), flow cytometry can be used to identify and quantify these cells
- This is key in many areas of research, such as understanding the effects of drugs on cancer cells or studying autoimmune diseases
4) Intracellular Cytokine Staining:
- This technique allows the detection of cytokines and other intracellular molecules within individual cells
5) Hematology:
- Flow cytometry is used in automated complete blood counts, reticulocyte counts, and the diagnosis and monitoring of hematological malignancies
6) Immunology:
- It’s used to study immune cell subsets, cytokine production, receptor expression, and a host of other immunological parameters
7) Microbiology:
- Flow cytometry can be used to study a variety of features of bacteria, including antibody binding, DNA content, and even bacterial cell size and granularity
8) Cell Sorting:
- Flow cytometers equipped with cell sorting capabilities (often called cell sorters) can be used to separate and collect cells of interest based on their specific light scattering and fluorescence characteristics
Discuss flow sorting
Flow sorting, or fluorescence-activated cell sorting (FACS), is a specialised type of flow cytometry
It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescent characteristics of each cell
1) Cell Preparation:
- Similar to standard flow cytometry, the cells must be in a liquid suspension and can be stained with fluorescent markers to tag specific components or characteristics
2) Flow:
- The cell suspension is introduced into the flow cytometer instrument, where it is focused into a single-cell stream
3) Interrogation:
- The cells pass through a laser beam, where light is scattered and fluorescence from the cells is collected by detectors
4) Decision Making:
- Each cell’s properties (size, complexity, and fluorescent characteristics) are analysed in real time by a computer
- Depending on the criteria set by the operator (such as the presence or intensity of a specific fluorescence), a decision is made on whether to sort a particular cell into a separate collection container
5) Sorting:
- The actual sorting of cells is achieved by applying an electrical charge to the droplet containing the cell as it is being formed. This charge can be either positive or negative, and it determines into which container the droplet (and thus the cell) will be deflected as it falls through an electrical field
- Uncharged droplets continue to fall straight down and are collected as the “waste” or “negative” population
6) Collection:
- The sorted cells are collected into separate containers for further analysis or experimentation
