Flow Cytometry: Applications

Question 1

Draw and label a diagram of the normal cell cycle.

Answer 1

Question 2

Describe cell cycle analysis using flow cytometry, and some commonly used fluorochromes.

Answer 2

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Question 3

Describe how flow cytometry can be used for assessment of cell viability, and some commonly used fluorochromes.

Answer 3

• Propidium iodide (PI) fluoresces when it binds to DNA
• PI can only cross the cell membrane of damaged or dead cells
• Thus PI can be used to distinguish between healthy and damaged/dead cells - i.e. it can be used to assess cell viability

Question 4

What is apoptosis?

Answer 4

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Question 5

Describe the techniques used to study apoptosis.

Answer 5

• Staining with the dye PI - cells fixed.
• Phosphatidyl serine can be detected by incubating the cells with fluorescein-labelled Annexin V and PI - FLAV binds to PS, which is only present on the extracellular membrane when a cell is undergoing apoptosis.
• Staining with 7-aminoactinomycin D - cells not fixed.

Question 6

Describe how flow cytometry can be used for the quantitation of apoptosis, and some commonly used fluorochromes.

Answer 6

• Populations of live, early apoptotic and late apoptotic/necrotic cells can be quantitated at the same time using Annexin-V-FITC and PI.
• These 3 cell populations can be visualised on a dot-plot: live cells will be negative for both dyes, apoptotic will be positive for Annexin-V but not PI, and dead will be posititve for both.

Question 7

Explain the reasons why different fluorochromes are used for cell cycle analysis, viability staining, and apoptosis quantitation.

Answer 7

• Cell cycle analysis - PI used to quantitate DNA
• Viability staining - cells that fluoresce with PI are damaged or dead
• Apoptosis quantitation - 3 methods: PI with fixed cells, Annexin V + PI (cells not fixed), 7-AAD (cells not fixed)
• Annexin V used to distinguish between early apoptotic, late apoptotic and dead cells

Question 8

List some clinical and research aplications of flow cytometry.

Answer 8

• Immunophenotyping of leukaemias & lymphomas
• Detection of MRD
• Stem cell enumeration
• CD4/CD8 in HIV
• Measurement of intracellular cytokines
• Study of cell cycle, viability & apoptosis
• Measurement of cell proliferation
• Assessment of transfection efficiency

Question 9

Outline the differences between flow cytometry and flow sorting.

Answer 9

• Flow cytometry used for comparing characteristics of different cell populations
• Flow sorting used to isolate highly purified (99% pure) populations of cells for analysis

Question 10

Describe the basic instrumentation of a flow sorter.

Answer 10

Question 11

Describe the principles of flow sorting.

Answer 11

• In flow sorting nozzle-tip vibrated at 1000s rpm
• This separates the stream containing cells into individual droplets which each contain one cell
• Electric charge is applied to the droplet containing the desired cells as they leave the stream
• Charged cells attracted to deflection plates and sorted into different tubes
• Flow sorting used to isolate purified cell populations

Question 12

Explain when fluorescence-activated cell sorting (flow sorting) is used and describe some of its applications.

Answer 12

• Diagnostics - specific cell populations isolated for analysis, e.g. cancer cells, CD4/CD8 in HIV
• Allows immunophenotyping
• Stem cell enumeration - isolating and counting stem cells

Question 13

What are the advantages of using 7-aminoactinomycin D (7-AAD) for quantitation of apoptotic cells?

Answer 13

• DNA-specific - intercalates in GC regions.
• Long emission wavelength - can easily be used with FITC and PE labelled Abs for simultaneous evaluation of DNA content and 2-colour immunofluorescence.