In June, the main focus was to familiarize myself with the background for the project - My progress introduction. This required an extensive literature search covering the role of IL-25 in allergic airway inflammation, and the relationship between mitogen-activated protein kinases (MAPKs) and asthma. At the same time, I also started to get familiar with the isolation procedure for B cells and other techniques that I might use in the project.
Based on literature review, the concentration of IL-25 used to achieve stimulation was in the broad range of 50ng/ml to 2µg/ml. After consulting my supervisor, I decided to first use 100ng/ml of IL-25 to stimulate the signalling pathway in stimulated B cells. Then the time-response of MAPKs (including P38 and P44/42) to IL-25 experiment was set up. The time intervals for p38 were 0, 5, 15, 30 minutes and one hour and the time intervals for p44/42 were 0, 1, 5, 15 and 30 minutes respectively. Flow cytometry was used to measure the MAPKs activation using phosphor-specific antibodies to p38 and p44/42. No phosphorated p38 was detected while a slight but not increase was detected for phosphated p44/42. For the second trial, the concentration of IL-25 was same as before, but I set up a positive control by using 10-8M PDB to stimulate MAPKs activation. The time intervals for this trial were 2 minutes and 5 minutes. A significant increase in the level of phophorated p44/42 was observed, but there was no change for IL-25 stimulation.
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Since the two trials did not work well, my supervisor advised me to use CD23 (the marker on the B cells, can serve as a low affinity receptor for IgE and involved in the regulation of IgE synthesis, and the CD23 suspected to down regulate IL-25 ) to determine the function of IL-25 in B cells. This set up involved dose-response studies of IL-25, so B cells were incubated for 24 hours with100ng/ml and 200ng/ml of IL-25. A significant change was not observed after staining the cells with anti-human CD23. I think the experiment failed because of my lack of experience in flow cytometry and staining procedures, so I will repeat this experiment again to get better results.
Since fresh human B cell preparation involves long time and considering my project timeline, I will use RAMOS B cell line to repeat the above mentioned experiment.