FC Flashcards
What is flow cytometry?
→Measuring 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 cytometry tell us about a cell?
→Its Relative Size
→Its Relative Granularity/Internal Complexity
→Its Relative Fluorescence Intensity
What proteins can be used to identify specific cell subsets?
→cytokine or chemokine
→metabolic proteins
→adhesion molecules
What are the methods of visualisation?
→Fluorescence Microscopy
→Flow Cytometry
What are the differences between the methods of visualisation?
→Microscopy- have to scan many fields but FC can analyse thousands of cells.
→FC can detect rare cells.
→FC quantitates the amount of fluorescence in each cell
What can the basics of flow cytometry be divided into?
→fluidics
→optics
→electronics
What are the basics of FC?
→Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer
What are the basic stems of FC?
light source→ flow chamber→ optical system→ light detectors→ computer
How are cells suspended in a single file?
→by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
→hydrodynamic focusing
What is hydrodynamic focusing?
→Introduction of a large volume into a small volume
→when two streams of fluids with different flow rates are running side-by-side and in the same direction into a flow cell, then a laminar flow is created
What is laminar flow?
→Sample fluid flows in a central core that does not mix with the sheath fluid
What is involved in the optics stage of FC?
→lasers
Describe lasering of the cells
→Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
→can provide from milliwatts to watts of light
What sort of frequency is the wavelength of light in the lasers?
→Single frequency
→coherent light
Which is more expensive? Air cooled units or water-cooled units?
→water-cooled units
What is the meaning of the forward light scatter?
→proportional to size
What is the meaning of the side scatter?
→granularity
What are dichroic filters?
→direct certain wavelengths of light one way, while deflecting other wavelengths
What are PMTs?
→amplifies the signal
→photomultiplier tubes
What is involved in the electronics part of FC?
→Processing of signals from detectors
→Analog-Digital Conversion