Cells from individual diaphragms were recovered following perfusion of blood from tissues, as described previously (22 (link)), and cultured ex vivo for 6 h with 250 ng/ml ionomycin (Sigma-Aldrich), 50 ng/ml PMA (Sigma-Aldrich), and 1 μg/ml brefeldin A (BD Pharmingen). After a 15 min incubation with Fc block (eBioscience) and 10% normal mouse serum, cells were incubated for 15 min with PE-Cy5-conjugated anti-CD4 (eBioscience). Samples were then treated with fixation/permeabilization buffer (eBioscience), and permeabilized cells were incubated for 1 h with PE-conjugated anti-IL-4 (eBioscience) or anti-IL-10 (eBioscience).
For dendritic cell phenotyping, cells from dLNs and diaphragms were incubated FITC-conjugated anti-CD11b, PE-conjugated anti-CD11c and Pacific blue-conjugated anti-MHCII. Data were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Tree Star).
For dendritic cell phenotyping, cells from dLNs and diaphragms were incubated FITC-conjugated anti-CD11b, PE-conjugated anti-CD11c and Pacific blue-conjugated anti-MHCII. Data were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Tree Star).
