What are the applications of flow cytometry?
Used for the analysis of cell cycle position by quantitation of cellular DNA
It is still the method of choice for fast, accurate determination of cell cycle distributions
What are univariate cell cycle methods?
In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
What is propidium iodine?
Most commonly used a fluorescent dye that undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane. It attaches PI to DNA. PI emits at 600-620nm.
How does PI measure the cell cycle?
The machine quantifies the no. of cells in each peak. The increase in PI intensity against cell count.
How does PI show cell viability?
- PI cannot normally cross the cell membrane but holes are punched in plasma membranes to allow PI to enter.
- If the PI penetrates the cell membrane, it is assumed to be damaged.
- Cells that are brightly fluorescent with the PI then, they are damaged or dead.
What is apoptosis?
Programmed cell death where the cell goes through a highly regulated process of “dying”.
How is apoptosis measured?
- Characteristics are the 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
Difference between apoptosis and necrosis
- In necrosis, small blebs form and the structure of the nucleus changes and during apoptosis, small blebs form.
- In necrosis, the blebs fuse and become larger, no organelles are located in the bleb whereas, in apoptosis, the nucleus begins to break apart, and the DNA breaks into small pieces. The organelles are also located in the blebs.
- In necrosis, the cell membrane ruptures and releases the cell’s content; the organelles are not functional. Whereas, in apoptosis, the cell breaks into several apoptotic bodies; the organelles are still functional.
What are the 3 ways of detecting apoptosis by flow cytometry?
- Need to punch holes into the membrane so by staining with the dye PI (cells fixed).
- Phosphatidylserine can be detected by incubating the cells with fluorescein-labeled Annexin V, and Pi (cells not fixed). The Annexin binds to PS to trade cells in apoptosis.
- By staining with 7-aminoactinomycin D (Cells not fixed)
What is the peak sub G0 thought to be? How was the peak determined?
Thought to be apoptotic cells but others think it might be debris. An agent was added to cause apoptosis and the PI. It shows overtime the sub-G0 peak increased.
What are the effects of PI and Annexin V-FITC on live, early apoptotic and late apoptotic/necrotic?
- Live: negative for PI and Annexin V-FITC as PI cannot enter as the membrane isn’t damaged and Annexin V cannot bind.
- Early Apoptotic: positive for Annexin V-FITC as PS switches but negative for PI as the membrane isn’t damaged.
- Dead: Positive for both
What is 7-aminoactinomycin D (7-AAD)?
- A fluorotic
- Similar emission to PI
- DNA-specific: intercalates in G-C regions
- Long emission wavelengths because it has lower wavelengths and can be run at the same time as FITC and PE.
- Laser is about 488 nm
- Emission at 680 nm
What are the applications of flow cytometry?
- Immunophenotyping of leukaemias and lymphomas
- Detection of MRD
- Stem cell enumeration
- CD4/CD8 in HIV
- Measurement of intracellular cytokines
- Study of the cell cycle, viability and apoptosis
- Measurement of cell proliferation
- Assessment of transfection efficiency
How are cells sorted?
- Cells introduced
- Laser hits as they move in a single file
- Cells express different immunofluorescence
What are cells like in pre-sort and post-sort?
Pre-sort
- Peaks are irregulated
Post-sort
- Peaks are tight and usually under one emission spectrum
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Cellular Growth Regulation53
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Cell Death and Cell Damage48
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Mechanisms of Oncogenesis60
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Oncogenes and Tumour Suppressor Genes52
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Tumour Angiogenesis, Invasion, and Metastasis37
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Primary Cell Culture Technqiues47
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Cell Culture Techniques46
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Microscopy32
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MRI and MRS71
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Ultrasonagraphy62
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X-ray, CT, PET41
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Flow Cytometry - Introduction35
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Flow Cytometry - Applications15
