Figure 4: Flow cytometric analysis of Tregs and quantitative real-time PCR analysis of Treg-associated molecules in Tregs isolated from CLN of sublingually treated mice with allergic rhinitis. (a) The frequency of CD4+CD25+ Tregs in spleen, CLN, NALT, and nasal passage of sublingually treated mice with allergic rhinitis were determined and calculated using a flow cytometer. (b) Messenger RNA expression of Foxp3, IL-10, and TGF-β in CD4+CD25+ Tregs isolated from CLN of sublingually treated mice was determined by quantitative real-time PCR analysis. The expression of each molecule was normalized to the expression of GAPD. Each data was expressed as a ratio relative to mean expression level in PBS-treated control mice. Data are representative of two separate experiments. Significance was evaluated by an unpaired t-test. and .