Pe labeled igg2a

Expression of the RAW264.7 cell-surface markers cluster of differentiation 206 (CD206; M2 marker) and C-C chemokine receptor type 7 (CCR7; M1 marker) was determined by flow cytometry. After 4 days of culture on samples, RAW264.7 cells were collected, centrifuged at 1,200 rpm for 5 min at 4°C, resuspended in PBS containing 1% bovine serum albumin (BSA) to block Fc-receptors for 30 min at room temperature, and incubated with phycoerythrin (PE)-conjugated anti-mouse CD206 and allophycocyanin (APC)-labeled anti-mouse CCR7 (eBioscience) antibodies for 1 h at room temperature in the dark. PE-labeled IgG2a and APC-labeled IgG2a (eBioscience) were used as negative controls. The cells were washed three times in PBS containing 1% BSA and transferred to FACS tubes (200 µL per tube) for determination using a Guava easyCyte™ HT flow cytometer (Millipore, Billerica, MA, USA); 5,000 events per tube were analyzed. Results were processed using guavaSoft 3.1.1 software.

For Foxp3 staining, whole blood samples were co-stained with anti-CD4-FITC and anti-CD25-PE (PC61.5). After fixation and permealization, cells were stained with APC-labeled anti-Foxp3 mAb (FJK-16s) (all mAbs were from e-Bioscience) as described (Chen et al., 2007 (link)). For IL-6, and IL-17 staining, 2 x 10⁶ spleen cells were stimulated with 10 ng/ml phorbol myristate acetate (PMA) (Sigma) and 10 ng/ml ionomycin (Sigma) in the presence of the Golgi inhibitor Brefeldin (Sigma) (10 μg/ml) for 4 hr. The cells were then stained with FITC-labeled anti-CD4 (GK 1.5, e-Bioscience) and PE-labeled anti-IL-6 (MP5-20F3, BD Pharmingen) or APC-labeled anti-IL-17 (eBio17B7, e-Bioscience) as described (Kappel et al., 2009 (link)). FITC-labeled anti-CD8 (53-6.7) and MHC class II (M5/114.15.2, e-Bioscience) were used for FACS analysis on T cells and BMSCs. PE-labeled IgG2a, FITC-labeled IgG1, APC-labeled IgG2a and IgG2a, κ were used as isotype controls (all from e-Bioscience). Cells were analyzed on a FACScan with Cellquest software (Beckton Dickinson). For the co-cultures with T cells and BMSCs, T cells were stimulated with Brefeldin (10 μg/ml) alone for 4 hr.