8. Flow Cytometry II Applications Flashcards
Cell cycle analysis methods.
- One of the earliest applications of flow cytometry was the analysis of cell cycle position by quantitation of cellular DNA.
- Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions.
Briefly describe how flow cytometry works
- In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
- Propidium iodide (PI) is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization (punch holes into the membrane) of the plasma membrane.
Describe how DNA content changes in a cell cycle. - link with the phases most of the cells are in. How are the cells quantified?
- Most cells are in the G0 or the G1 phase ~ 2N
- The least amount of cells are in the S phase
- There is slightly more cells in the G2 or M phase ~ 4N
- PI allows for quantitate the different proportions of cells in each phase
Describe how flow cytometry can be used for assessment of cell viability, and some commonly used fluorochromes
- PI cannot normally cross cell membrane
- If PI penetrates the cell membrane, it is assumed to be damaged
- Cells that are brightly fluorescent with the PI are damaged or dead
Expand on apoptosis.
• Programmed cell death, goes through highly regulated process of ‘dying’
• Characteristics:
- Condensation of chromatin material
- Blebbing nuclear material (bulging out/protruding, due to decoupling of cytoskeleton from structure)
- Often accompanied by internucleosomal degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis
Contrast the different events that lead to apoptosis (in a normal cell) and necrosis.
• APOPTOSIS
1) Small blebs form
2) The nucleus begins to break apart, and the DNA breaks into small pieces. The organelles are also located in the blebs
3) The cell breaks into several apoptotics bodies; the organelles are still functional
• NECROSIS
1) Small blebs form; the structure of the nucleus changes.
2) The blebs fuse and become larger; no organelles are located in the blebs.
3) The cell membrane ruptures and releases the cells’s content; the organelles are not functional.
What are the different detection methods for apoptosis?
1) By staining with the dye PI (cells fixed)
2) Phosphatidyl serine, can be detected by incubating the cells with fluorescein (FITC) labelled Annexin V, and PI (cells not fixed) ~ PI will then be detected inside the cell
3) By staining with 7-aminoactinomycin D (cells not fixed)
Expand on using PI to detect apoptosis.
- Fluorescence intensity is on the x-axis and the cell count is on the y-axis.
- Sub-G0 peak is not always seen – unreliable method ~ Some people say this peak is not really apoptotic cells, by just DNA fragments
- When cells are treated to induce apoptosis - the sub-G0 peak gets bigger over time
Expand on using PS to detect apoptosis.
- Early apoptosis: PS expressed outside of cell in apoptosis, add AnnexinV-FITC and it binds -> cell lights up green
- Late apoptotic/necrotic: AnnexinV-FITC can bind and PI can get in and so is positive in the cell -> positive in green and red
- Dot plot shows x-axis: annexinV-FITC and y-axis: PI -> live cells negative for both, dead cells (late apoptotic) positive for both, apoptotic cells light up for annexinV-FITC
Expand on using 7-Aminoactinomycin D to detect apoptosis.
- Excited with 488nm laser
- Emits at around 660nm (red)
- DNA-specific
- Long emission wavelength – can use green and yellow for surface staining at the same time (with FITC and PE labelled Ab for evaluation of DNA content and 2-colour immunofluorescence at the same time, using only 488nm laser to excite)
- Frees up other photomultiplier tubes on machine to do surface staining at the same time (to identify different populations)
- Graph showing FS/size (x-axis) and 7-AAD illumination intensity (y-axis: Live cells don’t take it up, apoptotic dimly lit, dead cells very bright
Expand on using the components of the apoptotic pathway
- Parts of apoptotic pathway can be used to quantitate what is going on (via flow cytometry)
- Antibodies for different caspase components -> quantitating different caspases -> using different colours in combination with surface staining (allows us to look at subpopulations of cells)
Examples of flow cytometry applications
- Immunophenotyping of leukaemias and lymphomas
- Detection of MRD (minimal residual disease)
- Stem cell quantitation
- CD4/CD8 in HIV
- Measurement of intracellular cytokines
- Study of cell cycle, variability and apoptosis
- Measurement of cell proliferation
- Assessment of transfection efficiency
Outline differences between flow cytometry and flow sorting
- Allows us to define a population of cells and purify (sort) them -> purification of cells
- Take them away from machine and grow them in culture for further investigation
Describe how the first part of a flow sorter works. (same as flow cytometer)
- Sample through central core
- Surrounded by sheath fluid that makes cells flow in a single file at centre (hydrodynamic focusing)
- Laser of single wavelength hits cells
- Light scatter or fluorescence emitted
- Information from photomultiplier quantified by computer
Describe how the second part of a flow sorter works. (different to a flow cytometer)
- Once data analysed by computer, wanted population of cells can be selected (e.g. cells in region 1 to right tube, region 2 in left tube, etc)
- Sort decision fed back into the machine to allow population sorting into tubes (can be 2 at a time)
- HOW??? Nozzle tip is vibrated so cell flowing in stream breaks off into droplets
- When wanted cell gets to end of nozzle, charge applied to the stream -> cell breaks off from stream in a charged droplet -> attracted by deflection plates into the tube for sorting
- Anything that doesn’t satisfy either wanted regions goes into waste
