Flow Cytometry Workshop Flashcards
Why is Flow cytometry used?
It is a high through put and high sensitivity method of identifying single cells of interest.
What fields use flow cytometry?
- Proteomics
- Cytomics
What are the roles of flow cytometry?
- Multi-colour surface staining
- Cell sorting or purification
- Expression cloning
What is expression cloning?
Enriching a very low frequency population and expanding them.
What are the different types of flow cytometers?
- Mobile to work in remote areas with remote populations like in Africa.
- Static which are very expensive and large.
- Multiple laser lines to detect more sub populations in one run.
Define flow cytometry
Measuring properties of cells in a fluid stream.
Define Fluorescence-Activated Cell Sorting
Sorting the cells based on the properties measured in the flow cytometry
What can you measure with a flow cytometer?
- Relative cell size and granularity
- Fluorescence emission when dyes bound to cells are excited by the laser
- Emission from many dyes at once for each cell
- Data from thousands of cells per second which allows you to see subtle changes
What happens in a flow cytometer?
- The flow cell focuses the cell suspension to make a single line of cells.
- A laser is shone at the cell line and we can get information about the cell from how the light scatters
- We can get additional information about the cell by using fluorescent dyes and measuring the wavelength of light emitted.
What does the forward scatter tell us?
The size of the cell
What does the side scatter tell us?
The shape and granularity of the cell
How is the scatter data visualised?
By converting the light into a voltage and then making a plot
What do the fluorescent dye wavelength emissions tell us?
- What dye is attached to the cell and therefore identify the cell
- How much of the dye there is
How are fluorescently labelled antibodies used in flow cytometry?
- To identify the phenotype of receptors present on the cells you are measuring
- Test large numbers of cells
- Measure multiple characteristics by using multiple labelled antibodies
- dissect complex cell populations
- define rare populations
- measure relative levels of antibody binding
How can a cell sorter be used in flow cytometry?
- It sorts cells of interest based on the fluorescent signals.
- An electrical charge is applied to the cell and this sorts them onto charged plates.
Flow cytometer data presentation: single parameters
On a histogram
Flow cytometer data presentation: Bivariate data
Displayed in 2 dimensions using dot plots, density or contour plots
Flow cytometer data presentation: 3 or more parameters
Displayed as sets of histogram or bivariate plots or using cluster analysis
How is gating used in flow cytometry?
- To detect rate cell populations like stem cells.
- To remove dead cells from the data
- To prevent false positives
- To identify any unwanted cells
What is MFI?
Mean fluorescence intensity which is the comparison of brightness of different cell populations or samples
What are the applications of flow cytometry?
- Analysis of surface or intracellular protein expression
- Resolution of complex populations
What are the applications of Flow sorting?
- Isolation of individual subpopulations from complex mixtures of cells
- Cloning specific cell populations
What are fluorescent tracking dyes used for?
Measuring cell proliferation
How do fluorescent tracking dyes work?
- They are cell permeable and covalently couple to intracellular proteins.
- Can identify up to 8 cell divisions as the signal halves with every division
What makes a flow cytometer a sorter?
The ability to select any population defined by a logical combination of regions and isolate this population from the sample.
How is flow cytometry used in gene expression studies?
- To purify cell populations before microarrays
- Post gene-chip-confirm protein expression using reporters
What are the uses of flow cytometry and nucleic acid stains?
The purpose of these is to measure the DNA content.
1. Cell cycle analysis
2. Measure ploidy
3. DNA probes
