Flow Cytometry- Introduction And Applications Flashcards
What is flow cytometry?
A technique that simultaneously measures several physical characteristics belonging to a single cell in suspension
What is the definition of flow cytometry?
Measuring the properties of cells in flow
What is flow sorting?
Sorting (separating) cells based on properties measured in flow
What is another name for flow sorting?
Fluorescence-activated cell sorting (FACS)
What can a flow cytometer tell us about a cell?
Relative size,
relative granularity/ internal complexity,
Relative fluorescence intensity
What can we measure with a flow cytometer?
Surface receptors
Intracellular cytokines/enzymes
Measure cell cycle viability and apoptosis
What are fluidics?
Cells in suspension flowing in a single file
How do fluidics work?
Inject a sample into a sheath fluid as it passes through a small hole
Simple fluid flows down a central core
What is hydrodynamic focusing?
Introduction of a large volume into a smaller volume
What are the steps in optical flow cytometry?
Illuminate fluid using a laser
Light scatters, emitting fluorescence
Fluorescence is collected and filtered
What is a laser line?
Single wavelength of light
What is the forward light scatter proportional to?
Size
What is a 90 degree light scatter proportional to?
Granularity
What can you do with a foward and 90degree light scatter?
Plot on a graph and identify the cell
How do you filter the light after scattering in optical cytometry?
Mirrors that separate out the light
What is the stokes shift?
The energy difference between the lowest energy peak of absorbance and the highest energy of emission
What are the names of the fluorochromes?
Fluorescein isothiocyanate (FITC)
Phycoerythin (PE)
Peridinin chlorophyll protein (PerCP)
What colour does FITC emit?
Green
What colour does PE emit?
Orange
What colour does PerCP emit?
Red
What frequency is FITC excited and emited at?
Excited at 488 and emits at 520
What frequency is PE excited and emitted at?
Excited at 488 and emits at 580
What frequency is PerCP excited and emitted at?
Excited at 488 and emitted at 620
What are the ideal samples for flow cytometry?
Peripheral blood Bone marrow Fine needle aspirate CSF and other fluids Fresh tissue
What are the methods of labelling in flow cytometry?
Direct and indirect
How does direct labelling in flow cytometry work?
Monoclonal antibodies are preconjugated to fluorochromes
How does indirect labelling in flow cytometry work?
Unconjugated monoclonal antibodies
How is flow cytometry data displayed?
On a histogram or a box plot
What is gating?
Drawing a ‘gate’ around areas of interest on a computer and then getting the computer to show just those areas in more detail
What is cellular DNA detected using?
Propidium iodide
Fluorescent dye that binds preferentially to DNA
What does propidium iodide require?
Permeabilisation of the plasma membrane
At what fluorescence does propidium iodide excite and emit?
Excitation at 488, emits at 620
How does propidium iodide work?
If it penetrates the cell membrane you can assume it to be damaged
Cells that are brightly fluorescent with it are damaged or dead
What are some detection methods for apoptosis?
Propidium iodide
Phosphatidyl serine
7-aminoactinomycin D
Where does propidium iodide peak?
Sub G0
Why is propidium iodide not very reliable?
May detect just cell fragments and not all cell types show the sub G0 peak
How can phosphatidyl serine be detected?
Incubating cells with a fluorescein labelled annexin V and propidium iodide
Where is phosphatidyl serine normally and in apoptosis?
Phosphatidyl serine is normally on the inside of the cell but flips to the outside of apoptotic cells
What does annexin V bind to?
Phosphatidyl serine when it is on the outside in apoptosis
When does annexin V show up?
Early and late stages of apoptosis
When does propidium iodide show up?
Late apoptosis and necrotic cells
At what fluorescence is 7-aminoactinomycin excited and emitted?
Excited at 488 and emitted at 660
Where does 7-aminoactinomycin intercalate?
G-C regions
How do cell sorters work?
When the cell of interest reaches the tip of the nozzle it vibrates
-> charges the cell so it is pulled into a tube
What are the applications of cell sorting?
Immunophenotyping of leukaemias and lymphomas Detection of MRD Stem cell enumeration CD4/CD8 in HIV Measurement of intracellular cytokines Study of cell cycle, viability and apoptosis Measurement of cell proliferation Assessment of transfection efficiency
