flow cytometry Flashcards
what is flow cytometry?
quantitative analysis of cells and cell systems suspended in a fluid system
what are the 4 components of a flow cytometry?
fluidics
optics
detectors
electronics
what is the principle of flow cytometry?
a beam of light directed onto stream of fluid containing cells
light scattered and fluorescence is emitted and detected by detectors positioned where the stream passes through light
converted to a voltage pulse
converted to a digital signal by the computer
what samples can be analysed on flow cytometry?
haematological
cell line
microbiological
what is side scatter proportional to?
the granularity or internal complexity of a cell
what is forward scatter proportional to?
the surface area or the size of a cell
what is the issue with using a combination of fluorochromes?
many fluorochromes have overlapping emission wavelengths
so the observed fluorescent signal detected by the flow cytometer may not be the actual signal displayed by the cell
why would you fix your cells prior to flow cytometry analysis?
you would fix your cells if you wish to look at the end point of a particular experiment so fixation would stop all processes at that point in time
why would you want to look at live cells?
to look at an event as it happens
what fixation methods are there?
paraformaldehyde and methanol
why might you permeabilise cells prior to flow cytometry?
cells are permeabilised when you want to stain intracellular structures or antigen epitopes
what can be used to permeabilise cells?
methanol or detergents which make holes in the bi-lipid layer
what are advantages of direct antibody staining?
faster as it is a one step process
% binding should be proportional to antigen being bound
cross reactivity is minimised
what are the disadvantages of direct antibody staining?
weak signal
limited flexibility
more expensive
what are the advantages of indirect antibody staining?
inexpensive
greater flexibility
amplified signal
