Flow Cytometry Flashcards
When the cytometry data is displayed on a scatter graph it is split into two axis. what are the names of each of these and what did they indicate ?
The X axis is know as the forward scatter and the Y axis is the Side scatter.
Forward scatter tell us about the cell size
Side scatter tells is about the internal complexity (Granularity).
Once you have identified the type of cell you have on the cytometry graph how do you go about finding what individual type of cell it it, e.g. Th vs Tc?
Via the use of fluorochromes.
Once the antibody binds to the cell surface receptor the fluorochrome binds to base.
When a light goes through the fluorochrome it fluoresces and this florescence is detected and converted into electrical signal.
The intensity of the florescence in proportional to its number of binding sites.
A plot can then be generated which will show the amounts of each CD number depending on the axis.
E.g CD4 on Y axis and CD3 on X axis.
What is the purpose of the fluidics system and how does it work ?
Allows single cells to be interrogated by the laser
Cells injected into a flowing stream of saline/distilled water and then compressed 1 cell diameter ready for laser.
what is the purpose of the Laser and optics sections ?
Lasers
Light source directed at the cells to observe the cells
Optics (mirrors):
Gathers the light after it has been directed at the cell and separate it into different wave lengths.
What is flow cytometry used for ?
Cell size
Cytoplasmic granularity
Cell surface receptors (phenotyping)
On a Cytometry graph how can you identify Lymphocytes, monocytes and neutrophils ?
Lymphocytes - Small forward scatter / low side scatter
Monocytes - Medium forwards scatter / Low side scatter
Neutrophils - Medium, large forward scatter / High side scatter.
What do the detectors do and how ?
Receive the light
Intensity is converted into an electronic signal (voltage)
Sent to computer software that visualises it as ‘dots’ – each dot is the signal received from one cell
What are the four parts of the Flow cytometry process ?
Fluidics system
Lasers
Optics
Detectors
What is Flow Cytometry and how many particles pass though the laser per second ?
Simultaneous measurements of multiple characteristics of a single cell within a fluid (e.g. saline or distilled water)
Up to 4000 particles per second pass through a laser in a stream of fluid
Each cell passes through one at a time
