Practical 5a: Flow Cytometry Flashcards
Define flow cytometry
A technique for counting, examining and sorting microscopic particles suspended in a stream of fluid
Explain how flow cytometry works
(3)
Physical characteristics of particles are measured while fluorescently labelled antibodies can be used to examine protein expression
A laser beam is directed at a hydro-dynamically focused stream of fluid containing the cells
A number of detectors measure properties such as forward scatter, side scatter and fluorescence at different wavelengths
What does flow cytometry use to fluorescently mark molecules?
Antibodies
Describe the stream used in flow cytometry
Hydro-dynamically focused stream
What do the detectors in flow cytometry measure?
Forward scatter
Side scatter
Fluorescence
What does forward scatter of light reflect?
The size of the particle
What does side scatter of light reflect
Complexity/granularity
Explain in your own words how the flow cytometer can count cells
(2)
The hydro-dynamically focused pressure of the liquid forces the cells into single file
The cells are marked with fluorescent antibodies which can be counted as they pass by the detector
What is the fluid in the flow cytometer called?
Sheath fluid
Give an example of how the flow cytometer is used to differentiate T and B lymphocytes from a sample of lymphocytes
Two antibody types are used:
- one is an antibody against CD4 (helper) which is fluorescently marked with FITC
- one is an antibody against CD8 (killer) which is fluorescently marked with PE
How many different fluorescent labels can be used in a flow cytometer?
Up to 17-18
How does the flow cytometer select the correct excitation wavelengths?
By using a set of filters
What scale are the dot plots created by flow cytometer in?
Logarithmic
What does each dot on a scatter plot indicate?
Each dot represents one event passing the laser
What CD would you use to detect wbcs?
Cd45
What CD would you use to detect T cells?
CD3
What CD would you use to detect B cells?
CD19/20
What CD would you use to detect helper T cells?
CD4
What CD would you use to detect killer T cells?
CD8
What CD would you use to detect neutrophils?
CD15
What CD would you use to detect monocytes?
CD14
What is CGD?
Chronic Granulomatous Disease
How do you diagnose CGD?
(2)
Patient’s cells are stained with dihydrorhodamine 123 which is non-fluorescent but when exposed to ROS it is converted into rhodamine which is fluorescent
Cells are stimulates to produce ROS and if they can they fluorescence
What is LAD?
Leucocyte adhesion deficiency
What happens in leucocyte adhesion deficiency?
(2)
Patient’s fail to express CD18, the B2 integrin chain
As a result, leucocyte can along the endothelial walls but cannot bind firmly and get into the inflamed tissues
How does flow cytometry diagnose leucocyte adhesion deficiency?
Flow cytometry can detect the expression of CD18 on white cells
What is XLA?
X-linked agammaglobulinemia
What happens in X-linked agammaglobulinemia?
(2)
A condition that manifests in males who have an inherited mutated copy of Brutons’ tyrosine kinase (Btk) on the X chromosome
It results in B cells not being produced
What mutation causes XLA?
A mutated copy of Brutons’ tyrosine kinase
