Flow Cytometry II: Applications Flashcards
Applications of flow cytometry
Cell Cycle Analysis
Measurement of Apoptosis
Cell cycle analysis in flow cytometry
univariate analysis of cellular DNA using a fluorochrome (most commonly Propidium Iodide) which undergoes an increase in fluorescence upon binding DNA
-PI checks for cell viability
What is required for Propidium Iodide to get into the cell and bind DNA?
It requires permeabilisation of the plasma membrane to get the PI into the cell
How is Propidium Iodide excited?
488nm laser excites the PI.
PI emits at about 600/620nm, giving a pink colour.
PI Flow Cytometric Assay
Used to evaluate cell viability:
- PI cannot normally cross the cell membrane
- If the PI penetrate the cell membrane, it is assumed to be damaged
- Cells that are brightly fluorescent with the PI are damaged or dead. allowing to quantitate live or damaged/dead cells.
In a viable living cell, PI cannot get in, therefore the cell stains negative.
Apoptosis
programmed cell death where the cell goes through a highly regulated process of “dying”
Characteristics of Apoptosis
· 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
Measurement of Apoptosis by Flow Cytometry
There are 3 ways of measuring apoptosis using flow cytometry:
1) By staining with the dye PI (cells fixed)
2) Phosphatidyl serine can be detected by incubating the cells with fluorescein-labelled Annexin V, and PI (cells not fixed)
- Phosphatidyl serine is normally inside the cell, but when the cell undergoes apoptosis, it flips onto the outside of the plasma membrane
- The fluorescein-labelled Annexin V binds to the phosphatidyl serine
- The PI allows us to measured live apoptotic and dead apoptotic cells
3) By staining with 7-aminoactinomycin D (cells not fixed)
Measuring Apoptosis: staining with the dye PI (cells fixed)
If we have apoptotic cells, we get a sub-G0 peak on a histogram, which is believed to be apoptotic cells. However, some people believe this peak is debris or apoptotic bodies and not cells undergoing apoptosis.
Another disadvantage is that cells that are undergoing apoptosis don’t always display this peak, therefore it is not a great reliable method for measuring apoptosis.
However, if we add an agent into the cells which initiates apoptosis, then we stain cells with PI at various time points, we can see how that G0 peak increases over time, allowing for the quantification of apoptotic cells.
Measuring Apoptosis: incubating the cells with fluorescein-labelled Annexin V, and PI (cells not fixed)
Live cell contains phosphatidyl serine on the inside of the cell.
A live cell is negative for both PI and AnnexinV-FITC. This is because PI can’t get in, and the AnnexinV-FITC can’t bind to phosphatidyl serine because it is on the inside of the cell.
An apoptotic cell in contrast has phosphatidyl serine on the outside of the cell, and so AnnexinV-FITC will bind to it and these cells will be positive for AnnexinV-FITC. However, PI cannot get in because the plasma membrane still isn’t damaged. These cells will therefore be positive green and negative red.
Then when the cells go further down the apoptotic pathway or become dead, PI can then get in, and these cells will then be positive for both (positive green and positive red). Therefore, we can differentiate between those 3 populations using those two stains at the same time.
Results of incubating the cells with fluorescein-labelled Annexin V, and PI (cells not fixed) on a dot plot
the cells with fluorescein-labelled Annexin V, and PI (cells not fixed) on a dot plot
· Live cells are negative for both AnnexinV-FITC and PI
· Apoptotic cells are positive for AnnexinV-FITC but negative for PI
· Dead cells are positive for both AnnexinV-FITC and PI
Measuring Apoptosis: staining with 7-aminoactinomycin D (cells not fixed)
This fluorochrome is excited by the 488nm laser and emits at about 660nm (similar to PI):
- DNA specific
- Long emission wavelength
Advantages of staining with 7-aminoactinomycin D (cells not fixed)
We can use this to measure live apoptotic cells just using this one stain, but because it has a long emission wavelength, we can also use it as the same time as other fluorochromes for other antigens:
-can be used with FITC & PE labelled Ab for simultaneous evaluation of DNA content and 2-colour fluorescence using only 488nm excitation
Results of staining with 7-aminoactinomycin D (cells not fixed) on a dot plot
· Live cells are negative
· Apoptotic cells are dim
· Dead cells are bright
Other than apoptotic cells, what else can we measure in the apoptotic pathway to quantify apoptosis?
we can also measure caspases and other different parts of the apoptotic pathway and quantitate them
What is the difference between cell sorting and flow cytometry?
The difference is that the nozzle tip is vibrated so that the cells stay in flow for a certain time, then they break off into droplets from that vibration.
When the laser is interrogating the cells, we can tell the machine that when it sees a cell satisfying a specific region, it needs to wait until that cell is in that final drop (last cell in the stream). The machine then charges that stream and the drop breaks off. The drop that we want containing the cell we want is charged, then, that drop is pulled because of the deflection plates into tubes/plates/microscope slides etc. The minute that drop breaks off, the charge is taken off the stream until it sees another cell that satisfies that region. Again, it waits until it gets to the last bit, it charges, drop breaks off, charge comes off. This is happening thousands of times per second, calculating how long it has to wait before it charges.
