Flow cytometry Flashcards
What is fluorescence?
Light of a particular wavelength hits molecules and excites the electrons. The energy is unstable, and electrons collapse back, emitting light.
The wavelength of light that goes in is different from the wavelength of light emitted.
How is flow cytometry linked to fluorescence?
Flow cytometry is dependent upon fluorescent molecules.
Different fluorescent molecules may be excited by different wavelengths of light and emit different wavelengths.
How is flow cytometry linked to antibodies?
A lot of flow cytometry involves antibodies.
But antibodies are not needed for cytometry.
What is a flow cytometer?
A machine that analyses particles (cells) in suspension.
It counts the particles.
Tells you how big/ granular they are.
Detects fluorescent markers on the particles.
What information can flow cytometry give you?
What type of cells - how many and the markers they express.
What the cells do.
Quantification of rare populations.
Track changes over time.
Break down heterogenous populations of cells into individual populations.
What does flow cytometry rely on?
The cells must be in suspension.
The particles must be able to made fluorescent, through antibodies, nucleic acid probes or chemicals.
What are the limitations of flow cytometry?
Cannot give information about:
The cell location in the original sample, as in suspension.
What other cells or features the cells are near.
Information about a single cell, only populations.
What type of cells can be studied using flow cytometry?
Normally - eukaryotic and bacterial cells.
With better machines:
Organelles, chromosomes, viruses, organisms - drosophila embryos.
As long as the particles are in suspension.
What are the key features of flow cytometers?
Lasers - source of light.
Cells in single file, go through laser 1 by 1, by laminar flow.
Laminar flow - takes cells through fluid at faster pace so forces them in single file.
Detectors, see a range of light, turn into electricity.
Mirrors separate wavelengths of light.
Computer for data in real time.
What is forward scatter?
Laser is shone through fluid, when it enters the cell it refracts and the light beam becomes wider, and hits the detector.
There is a shield on the detector so that the detector doesn’t burn out.
Light only hits the detector when it is refracted - forward scatter.
How does the cell change the detection?
A larger cell produces a larger beam of light, so more light hits the detector and there is a larger electronic signal.
The signal is converted to voltage, so the bigger the voltage the bigger number.
What is side scatter?
Light bounces off stuff (mitochondria, nucleus, vesicles) in cell and scatters sideways, and is then detected by a side scatter detector.
Detects granularity - how much stuff is in the cell.
How does granularity change?
When there are fewer organelles there is less side scatter, and it is less granular.
The wavelength of light detected does not change.
It gives information about how active the cells are.
How can data from size and granularity be combined?
Every dot is a cell/event going through the flow cytometer.
Can see different populations of cells.
1st population large and granular. Macrophages.
3rd population, small and not granular, T and B cells
2nd population medium size and granularity. Monocytes.
See picture.
How are fluorescent antibodies used in flow cytometry?
Cells can be identified through the molecules on their outside and inside.
Add a different fluorescent molecule on each antibody.
Antibodies will stick to their unique molecule.
You will detect the fluorescence and be able to detect that cell.
How can T cells be detected in flow cytometry?
All T cells have a T cell receptor.
Helper T cells have CD4, cytotoxic T cells have CD8.
Use antibodies to the T cell receptor to detect T cells, then use antibodies to CD4 and CD8.
If not a T cell, nothing will bind to it.
How can different antibodies be used in flow cytometry?
How can different antibodies be used in flow cytometry?
How does fluorescence detection change in flow cytometry?
More antibodies or fluorescent molecules bound to the cell, the brighter the cell, more light emitted, so bigger voltage at the end.
How can fluorescence data be used to identify cells?
See picture
There will be 4 boxes - 2 single positives, 1 double positive, and 1 double negative.
As you go along the axis the signal is larger - the cell gets brighter.
How are the different markers in flow cytometry combined?
All the data on size, granularity and fluorescence is collected simultaneously.
The data is identified sequentially e.g. identify the right size, then broad markers then specific ones.
Regions are used to select cells and analyse them further.
See picture
What are the uses of flow cytometry?
Measures changes
Measures proliferation
Measures cell death
How does flow cytometry measure changes?
Decreasing cell numbers - through migration out or cell death.
Increasing cell numbers - through migration in or cell proliferation.
Flow cytometry gives quick data about if the cells are doing this.
How can a flow cytometer measure proliferation?
Using a dye that can enter cells but not leave.
e.g. carboxyfluorescein succinimidyl ester (CFSE).
When cells divide, the dye is divided between them, as they divide fluorescence divides.
How can flow cytometry measure cell death?
Normal cell has lipid bilayer, different lipids inside and outside, and DNA in nucleus.
Cell begins flipping membrane - phosphatidyl serine on outside, and annexin V binds to it.
Membrane becomes permeable, cells can be stained with DNA binding dye e.g. propidium iodide, which shows which cells are dying because the dye would not usually be able to bind.
What is FACS?
Fluorescence activated cell sorting.
When cell sorting you tell the machine what population of cells you are interested in and then it separates them out for collection.
Cells are separated by charge - negative go to positive plate.
The cells can then be used for culture, sequencing, genetic modification etc.
What is high parameter flow cytometry?
There are only 18 parameters, so for high parameter, use mass spectrometer.
e.g. blood from a baby with not a lot of blood to give.
Instead of fluorescence, uses metallic elements conjugated to the antibody.
Measures atomic weight, based on metals going through.
What are the pros of flow cytometry?
Lots of data
Takes hours (less than western blotting or immunohistochemistry).
Phenotype and quantify rare populations of cells.
Track changes with time.
Break down heterogenous populations of cells into individual populations.
What are the cons of flow cytometry?
No anatomical data.
Can be difficult to get cells into a solution.
Can only look at populations, not single cells.
From the data above what are the differences between CD4 and CD8 cells during the immune response?
See picture of data
The CD8 and CD4 cells before the experiment are fairly similar.
During the immune response more of the CD4 cells are producing IL-2 than the CD8s (bottom right). CD8s produce more IFNy than the CD4s (top left). Top right – production of both IFNy and IL-2.
- Can you think of any other ways to detect IL2 or IFN?
ELISA - cytokines such as IL2 and IFNy can be detected in solution such as blood plasma.
Or in suspensions of cells using ELISPOT.
Might look at mRNA as a proxy for the proteins using RTPCR.
Flow cytometry will give additional information such as specific cell population producing the cytokine whereas ELISA will better allow you to know the total quantity of the cytokine produced.
