Flow Cytometry - Introduction and applications Flashcards
Explain what is flow cytometry
- Measuring properties of cells in flow
- Sorting (separating) cells based on properties measured in flow
- Also called Fluorescence-Activated Cell Sorting (FACS)
What can flow cytometry tell us about cells
- Its Relative Size
- Its Relative Granularity/Internal Complexity
- Its Relative Fluorescence Intensity
List the 2 methods of visualisation
- Fluorescence Microscopy
- Flow Cytometry
What are the components involved in flow cytometry
- Light source
- Flow chamber
- Optical system
- Light detectors
- Computers
Explain the fluidics portion of the system
- Need to have cells in suspension flow in single file
- Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
- 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
What does each of the laser scatters show
- Forward light scatter proportional to size
- 90° Light scatter proportional to granularity
Explain channel layout for laser-based flow cytometry
- laser light is filtered by dichroic filters to block other wavelengths
- 3 different colours are used to look for 3 different antibodies
how are the signals from detectors processed
- analog-digital conversion of signals
Define fluorescence and what is stokes shift
- fluorescence is the emission of energy at a different wavelength than what it absorbed
- The energy difference between the lowest energy peak of absorbence and the highest energy of emission
List some fluorochromes and what colour they emit
Fluorescein isothiocyanate (FITC) - GREEN
Phycoerthrin (PE) - ORANGE
Peridinin Chlorophyll Protein (PerCP) - RED
What are the methods of labelling
DIRECT - Monoclonal antibodies (MoAbs) conjugated to fluorochromes
INDIRECT - Unconjugated MoAbs and secondary antibodies conjugated to fluorochromes
How can data from flow cytometry be displayed
- Histograms
- Dot Plot
Explain how we can analyse data from flow cytometry
- Select a population of interest and apply new analysis parameters to it
- can be done to either box plots or histograms
What are cell cycle methods
- 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
How do Propidium iodide assays work
- PI cannot normally cross the cell membrane
- If the PI penetrates the cell membrane, it is assumed to be damaged
- Cells that are brightly fluorescent with the PI are damaged or dead
how could we measure apoptosis?
- Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
- Characteristics are condensation of the chromatin material
- Blebbing of nuclear material
- Often accompanied by internucleosomal degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis
What are the methods for detecting apoptosis
- By staining with the dye PI (cells fixed)
- Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
- By staining with 7-aminoactinomycin D (cells not fixed)
How does 7-aminoactinomycin (7-AAD)
Ex: ~488 nm
Em: ~660 nm
DNA-specific:
intercalates in G-C regions
long emission wavelength:
with FITC & PE labelled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex
Explain how we can sort the samples as they are being examined
The machine vibrates and separates cells by charging them and deflecting them into tubes. the sorting is based on parameters set by the user.
What are the applications of using flow cytometery
- 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