I conducted an experiment with the Ramos cell line to analyze CD23 and its signaling pathway. The experimental setup I used for CD23 is similar to the one I used previously for human B cells. Compared with the nonstimulated cells, the level of CD23 expression in stimulated Ramose cells decreased, and as the concentration of IL-25 increased from 100 to 300 ng/ml, the CD23 expression level decreased successively.
Because the baseline in nonstimulated Ramos cells was quite high, I cannot identify the exact trend of CD23 expression. Then the time response of P44/42 to IL-25 experiment was set up.The time intervals were 0, 1, 5, and 15 min. No phosphorated p44/p42 was detected, whereas there was a slight phosphoration (for clarification: please check if the word used fit the meaning) in the first minute. The results showed that the concentration level of the second antibody was quite high and needs to be adjusted by series titration.
After conducting the titration experiment for the second antibody, 0. 3 ng of the second antibody with a concentration value of 0. 009 µg/µl and blocked by IVIG was needed for staining the cells.Because of the increase detected in the first minute, I repeated the experiment for P44/42 but in dose response, where the concentrations of IL-25 were 100, 200, 300, and 400 ng/ml, respectively.
On the other hand, the experiment in dose response was also set up for p38, where the concentrations of IL-25 were 50, 100, and 200 ng/ml, respectively. The p38 phosphoration was measured after 5 min of incubation. There was no increase detected for phosphorated p44/42 as well as for p38. Then, fresh B cells were used to conduct the same experiment for CD23 which was set up on the Ramos cell line.The result showed that there was great increase in CD23 expression level after B cells were stimulated by IL-4 and CD40.
After the addition of IL-25, the amount of CD23 was increased even further whereas the dose response of IL-25 seems not to effectively influence the CD23 expression level. This experiment was repeated once, and results were reproducible. In addition, the new experimental setup was also applied for CD23 by adding IL-4, CD40, and IL-25 together in the first day and measuring the amount of CD23 after 72 hr of incubation.The first trial showed the same trend as the one with 48 hr of incubation, but the result from the second trial was not repeatable because, after adding IL-25, the amount of CD23 decreased, and as the concentration of IL-25 increased, the amount of CD23 increased.
Thus, I plan to repeat the experiment of CD23 incubated in 48 hr, but a small amount of IL-25 would be used, and for 72 hr of incubation, B cells had to be stimulated by IL-4 and CD40 first and then IL-25 would be added and incubated for 72 hr in total.
