I´m performing DTH assays in mice. I usually immunize with 100 ug OVA in complete Freund´s adjuvant (1:1 mixture) by a subcutaneous injection, wait for two weeks and then inject 100 ug heat-aggregated OVA in 50 ul in one of the footpads. I also inject the same volume of PBS in the other footpad to control for the unspecific swelling response. There is commonly up to 0.1 mm swelling in the control footpad and at most 0.3 mm swelling in the antigen-loaded footpad. I was hoping to obtain more swelling or at least more reproducible reactions so I don´t have to work with many mice per treatment group. Has anyone tried using 10- to 100-fold higher doses of antigen for the DTH reaction?
Hi Jeremias,
Have you found the optimal dose for OVA induced DTH? We also want to do some research using this model.
Thanks
I'm currently using 100 ug of heat-aggregated OVA in 50 ul of PBS. I found out that the trick is to have a good CFA emulsion, otherwise you get reduced swelling responses. We are getting about 0.5 mm increase at 48 h. Hope this helps.
I saw you do the challenge 2 weeks later. Will it be better if you shorten it to be 7 days or 6 days? In my opinion, specific CD8 T cells will be triggered in 7 days. Maybe you wait too long. Just for your information.
You're absolutely right. We have shortened the interval to 7 days and we still get significant swelling responses. We also noticed that OVA-induced swelling reaches its peak at 48 h whereas saline-induced swelling decreases considerably at this timepoint. At 24 h we still have considerable non-specific swelling perhaps due to the trauma of the injection in the control footpad, which obscures the antigen-specific response that we are trying to detect.
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