Pe labelled anti foxp3

Aliquots of 10⁵ spleen cells/100 μL of staining buffer per well were incubated each with 1 μg of purified anti-CD16/CD32 for 20 min in the dark in order to block nonspecific binding of antibodies to the FcγIII and FcγII receptors. Subsequently these cells were stained with surface marker separately for 15 min with 1 μg of PE-Cy5-labelled anti-CD4 (GK1.5, eBioscience, San Diego, USA). For intracellular staining, spleen cells were first incubated with Inside Fix for 20 mins at room temperature and then stained with PE-labelled anti-Foxp3 (BD Pharmingen, Palo. Alto, CA, USA) in Inside Perm for 15 min in the dark. Corresponding PE-labelled rat IgG2a (kappa chain) was used as isotype control. Cells resuspended in 300 μL of buffer (0.15 M NaCl, 1 mM NaH₂PO₄ H₂O, 10 mM Na₂HPO₄ 2H₂O, and 3 mM NaN₃) were analysed in a flow cytometer (Becton Dickinson, Heidelberg, Germany) using the corresponding CELL QUEST software.

mDCs-2D and mDCs-3D derived from C57BL/6 mice were harvested as described previously and injected (3 × 10⁶ cells/mouse) into the C57BL/6 recipient mouse abdomen on days −6, −4, and 0. Then, splenocytes isolated from BALB/c mice were injected into the dorsal subcutaneous tissue of C57BL/6 recipient mice (1 × 10⁷ cells/mouse) on days 0 and 3. On day 7, C57BL/6 recipient mice were challenged by injection of BALB/c splenocytes into the right hind footpad (1 × 10⁷ cells/mouse). The left hind footpad of each recipient mouse was injected with the same amount of normal saline (NS) as a control. The negative control mice were injected with the same amount of NS. At 24 h after injection, the footpad thickness was measured using a micrometer. The extent of footpad swelling was measured by subtracting the baseline thickness of the left footpad from the thickness of the right footpad. The peripheral blood of experimental mice was collected to determine the levels of IL-10 and TGF-β1 using ELISA kits.
To determine the proportion of Treg cells, the splenocytes of experimental mice were isolated using Ficoll, and stained with FITC-labelled anti-CD4 (BD Pharmingen), APC-labelled anti-CD25 (BD Pharmingen), and PE-labelled anti-Foxp3 (BD Pharmingen). The proportion of Treg cells was analysed with BD FACSDiva Software v6.1.3.