Workshop 1

• Flow cytometry allows acquisition of multiple data sets simultaneously
• Blood sample can be stained for neutrophils, monocytes, T cells, B cells etc and multiple cell surface/intracellular markers can be assessed simultaneously
• Flow cytometry acquires data from 1000s/10000s of cells allowing generation of robust data
• Flow cytometry is used more in basic science research in a low throughput setting
• Cytokine bead array uses beads of known size/fluorescence, each capture bead has distinct fluorescence and is coated with a specific antibody
• Cytokine bead array can measure up to 30 analytes simultaneously using 50uL of sample
• Basics of flow cytometry involve cells being stained with fluorescently labelled antibodies, placed into the flow cytometer, passing through a laser one at a time, and the laser light being scattered in a forward and side direction
• In flow cytometry, the laser excites fluorophore which emits a particular wavelength, detected by the machine, and results can be seen on a computer
• In a typical dot plot of human blood, each dot represents a cell, forward scatter (FSC) indicates the size of the cell, and side scatter (SSC) indicates the granularity of the cell
• Neutrophils are larger cells with high granularity, monocytes are slightly smaller than neutrophils and less granular, and lymphocytes are the smallest leukocytes and least dense
• Neutrophils are identified by CD66b which is exclusively expressed on neutrophils
• T cells are identified by CD3+, CD4+, and CD8+
• Data analysis in flow cytometry involves selecting cell populations of interest, creating histograms, and using median fluorescence intensity (MFI) to compare expression levels
• In the mouse peritonitis model, an inflammatory response is elicited by injecting pathogens or irritants into the peritoneal cavity, leading to leukocyte trafficking and collection of peritoneal fluid for analysis
• Advantages of the mouse peritonitis model include mild to moderate severity, acquisition of large samples, and investigation of resolution pathways
• In an experiment testing a novel anti-inflammatory drug using the peritonitis and flow cytometry techniques, the drug is administered orally to the mouse, an inflammatory response is elicited by injecting an inflammatory stimulus, and peritoneal cells and lavage fluid are collected for analysis
• To test if an antibody is specific and binds to its intended target, negative and positive controls are needed, and antibody titration is performed to determine the appropriate concentration to use