Flow cytometry Flashcards
What is flow cytometry and how is it done?
Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension. It is done by light scatter and fluorescence.
What is flow sorting?
Separating cells based on properties measured in flow, also known as fluorescence, activated cell sorting (FACS).
What can a flow cytometer tell us about a cell (3 points)?
Relative size
Relative granularity/internal complexity
Its relative fluorescence intensity
Key differences between flow cytometry and fluorescence microscopy?
They differ in aspects of locating cellular components, the volume of cells they can handle, and sample preparation prior to analysis.
Fluorescence microscopy can show how component is distributed in the cell, it can also show whether concentration changes temporarily or remains static.
Flow cytometry cannot usually specify the location of the of the component in the cell.
Flow cytometry can handle larger volume and can use fluorescence properties to distinguish and sort cells.
The flow of the sample through the instrumentation?
Light source -> flow chamber -> optical system -> light detector -> Computer.
What are the basics of flow cytometry?
Fluidics, optics, and electronics
Cells in suspension and flow in a single file through an illuminated volume where they scatter light and emit fluorescence that is Collected, filtered, and Converted to digital values, stored in computer.
How is cells in suspension flow accomplished in fluidics stage of flow cytometry?
By injecting sample into a sheath fluid as it passes through a small orifices
What is laminar flow?
Sample fluid flows in a central core that does not mic with the sheath fluid
What is hydrodynamic focusing?
Introduction of a large volume into a small volume
What is FSC and what is it proportional to?
Forward light scatter, proportional to size
What is SSC and what is it proportional to?
90-degree light scatter proportional to granularity
What is fluorescence/ stroke shift?
is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
what are 3 common fluorochromes and what wavelength do they excite and emit?
FITC - excited at 488 and emits at 520 (green)
PE- excited at 488 emits 580 (orange)
PerCP- excited at 488 emits at 620 (red)
Why are filters and mirrors important?
Filters and mirrors essential in removing the overlap in the emission spectrum so that we can analyse data from 3 fluorochrome all together.
What are the common sources of cells for flow cytometry (5 points)
Peripheral blood Bone marrow Fine needle aspirate CSF and other fluids Fresh tissue
What is direct and indirect fluorescence labelling?
For direct immunofluorescence, the antibody binding the epitope is labelled with fluorophores (green). For indirect or secondary detection, the primary antibody binds the epitope and a fluorophore-labelled secondary antibody (purple) that has specificity for the primary antibody binds to it.
2 common data plot?
Histogram and dot plot
How can we use Propidium iodide to check for cell viability?
PI cannot normally cross cell membrane so if PI penetrates the cell membrane, it is assumed to be damaged
What are the 3 detection methods for apoptosis?
Staining with dye PI
Phosptidyl serine, fluroscein-labelled annexin V and PI
Staining with 7-aminoacinomycin D
How does Phosptidyl serine, fluroscein-labelled annexin V and PI work?
Phosphatidyl serine normally sits in the inside of the cell but during apoptosis it is flipped and expressed on the cell surface, thus annexin V- FITC is able to bind and make the membrane permeable to allow PI in to the cell.
List some application of flow cytometry and FACS
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
