I want to know if a specific antigen stimulates host immunity in vivo. So, I thought I can culture mouse splenic cells with this antigen for 2 days. Then, I collect the cells and I do flowcytometry for CD3+CD96 to know if T cells were activated upon second exposure.
Do you think this may work? any other suggestions to answer this question?
Thanks
Dear Reham Ajina,
What is your experiment? What kind of analysis you are doing, why you need to check activation of Total T cells?
If you want to study Th1 immune response, then I will suggest study cytokines IFNγ, IL2 and TNF α in CD3 gated CD4 and CD8 cells. If you want to explore a Th2 response, you can check IL4 or accordingly you can choose any cytokine. We usually stimulate splenocytes for 18-20 hours.
You can also do cytokine ELISA in total splenocytes cell culture medium, with or without your antigen.
The following article from our group may be useful; please check this out:
https://www.researchgate.net/publication/310838911_Generation_of_protective_immunity_against_Orientia_tsutsugamushi_infection_by_immunization_with_a_zinc_oxide_nanoparticle_combined_with_ScaA_antigen
All the best for your experiments.
Article Generation of protective immunity against Orientia tsutsugam...
Thank you Prashant Sharma for your response.
I need to confirm that my antigen of interest stimulates the host immunity. Previously, I injected tumor cells that express this antigen into mice and the tumors were rejected in WT mice but not in SCID mice. However, further validation is needed. Also, I did measure IgG1 levels in the serum of these mice and it was significantly different between the groups. Actually, I did measure IL2 secretion by ELISA but the result of the control was confusing. It seems that the control cells cultured without the antigen secreted higher levels of IL2 than with the antigen. I expected the levels of IL2 in the control group to be the same not to decrease. I did not have any interpretation other than that the ELISA was too sensitive and not very accurate. Therefore, I decided to test the same question in a different way, but knot sure how to do so.
Thanks
Dear Reham Ajina,
If you desire to examine the tumor regression, you should check Th1 and CTL response.
IgG1 Elisa is for a TH2 response which is mainly humoral immune response, Even if you want to check Elisa for Th1, I will suggest you to with IgG2c which is also there in my paper which I have linked in my last comment.
As far as ELISA for cytokine is concerned, please also check IFNγ and TNFα with IL2.
The best path would be to check FACS with a set of CD3, CD4, CD8, and IFNγ/ TNFα but if you don’t want, then Elisa is too beneficial.
I am not sure which cytokine Elisa kit you are using, please determine the standard value before starting the Elisa. Sometimes the values are excessively high; then you need to dilute your sample.
Some other significant detail is the “antigen stimulation time”, in your case, it seems stimulation is not executed in good order, you likewise require to optimize the stimulation time. One more thing always sets a positive control PMA/ Ionomycin so that you can compare your antigen stimulation.
Hope this little information can help you.
All the best