Flow Cytometry - Applications Flashcards
Describe the use of flow cytometry for cell cycle analysis over time
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.
Describe use of a dye in cell cycle analysis
Cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
Propidium iodide is most commonly used.
It undergoes a dramatic increase in fluorescence upon binding DNA.
It requires permeabilization of the plasma membrane.
Describe the potassium iodide assay
How the assay works:
PI cannot normally cross the cell membrane
If the PI penetrates the cell membrane, it is assumed to be damaged
Cells that are brightly fluorescent with the PI are damaged or dead
Apoptosis definition
Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
Apoptosis - characteristics
Characteristics are 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
Compare necrosis w/apoptosis
Blebs form, nucleus changes
Necrosis - blebs fuse = larger + no organelles in blebs
- CM ruptures, cell content released + organelles not functional
Apoptosis - nucleus/DNA break - organelles in blebs
- Cell into apoptotic bodies, organelles functional
Detection Methods for Apoptosis
By staining with the dye PI (cells fixed)
Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
By staining with 7-aminoactinomycin D (cells not fixed)
Applications - list
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
Flow cytometry and flow sorting - process comparison
Flow cytometry cell sorters have a collection system unlike flow cytometry analyzers.
Sample is injected into a stream of sheath fluid that passes through the flow cell and laser intercepts.
Stream carries the cell through a vibrating nozzle, disturbance in the stream = a droplet containing ideally one cell
An electrical charging ring is placed just at the point where the stream breaks into droplets
Also on the ring based immediately prior to fluorescence intensity being measured
Opposite charge is trapped on the droplet as it breaks from the stream.
Charged droplets fall through an electrostatic deflection system that diverts droplets into containers based upon their charge
