Flow Cytometry Flashcards
What is flow cytometry?
A technique which simultaneously measures several physical characteristics belonging to a single cell in suspension.
OR
Measuring properties of cells in flow
What is flow sorting?
Sorting cells based on properties measured in flow
Allows us to isolate very rare cells so we can do further analysis on them
What can a flow cytometer tell us about a cell?
- It’s relative size
- It’s relative granularity/internal complexity
- It’s relative fluorescence intensity
What are the two methods of visualisation?
Fluorescence microscopy
Flow cytometry - can be used to quantify rare cells and more accurately
What are the 3 stages of flow cytometry?
Fluidics
Optics
Electronics
What occurs during the fluidics stage of flow cytometry?
Cells in suspension
Flow in single file through a illuminated volume
Accomplished by injecting sample into sheath fluid as it passes through a smooth office
When sample fluid flows in a central core that does not mix with sheath fluid - laminar flow
The introduction of a large volume to a small volume - hydrodynamic focusing
What occurs in the optics part of flow cytometry?
Laser fired at cells
The cells scatter light and emit fluorescence that is collected and filtered and converted to digital values
Lasers:
- single wavelengths of light
- can be inexpensive, air cooled units, or expensive water cooled units
- provide coherent light
What occurs in electronics stage of flow cytometry?
Digital values stored on a computer
Label a simple diagram of a flow cytometer
Label a simple diagram of the channel layout in a flow cytometer
What is stroke shift?
The energy difference between the lowest energy peak of absorbance and the highest energy of emission.
Give examples of the fluorochromes and they’re respective dyes
Fluorescein isothiocyanate (FITC) - Green
Phycoerythrin. (PE) - Orange
Peridinin chlorophyll protein (PerCP) - Red
What is fluorescence?
When laser hits a fluorochrome and it’s excited at one wave length and then when it goes back to its unexpired state it emits fluorescence at a higher wavelength
Explain why combinations of fluorochromes can be used together?
Excited by the same laser but emit at different wave lengths
The filters and mirrors help to reduce overlap of light so we can analyse data from multiple fluorochromes together.
What are ideal samples to use for flow cytometry?
Single cells in suspension
- bone marrow
- peripheral blood
- fine needs aspirate
- CSF
- fresh tissue
What are the two ways of labelling cell surface proteins?
Direct: monoclonal antibodies (MoAbs) are pre-conjugated to fluorochromes
-uses one florescently labelled antibody to label cell
Indirect: un-cojugated MoAbs
- uses two antibody’s, with one being florescently labelled to label cell
What are the two most common ways of representing the data of flow cytometry?
Histogram
-one dimensional display
Dot plot
- two dimensional (can analyse 2 parameters at the same time)
What is gating?
Using our computer we can draw a gate over a population that we want to look at further on the basis of they’re florescence
- can further analyse by only showing cells in a certain parameter
How is flow photometry data analysed?
What is the simplest univariate cell cycle method?
Cellular DNA is detected using a fluorescent dye that binds preferably to DNA
Protium iodide is most commonly used
But it required permiabilization of the plasma membrane
Describe how a PI assay works
PI cannot normally cross 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
What are some characteristic of apoptosis?
Condensation of chromatin
Blabbing of nuclear material
Internucleosomal degradation of DNA
What are 3 detection methods for apoptosis?
By staining with the PI
Phosphatidyl serine, can be detected by incubating the cells with fluorescent labeled annexin V and PI
By staining with 7-aminoactinmycin D
What is 7-AAD?
Excitation: 488nm
Emission: 660nm
DNA-specific
Long emission wavelength
What are the applications of flow cytometry?
- immunophenotyping of leukaemia and lumphomas
- detection of MRD
- stem cell enumeration
- CD4/CD8 in HIV
- measurement of intracellular cytokines
- measurement of cell proliferation
- study of cell cycle and apoptosis
- asses,ent of transfection efficiency
