Anti il 10 apc

Flow cytometric analysis of B cell populations was performed using our previously described gating strategies8 (link)15 (link)16 (link)17 (link). Following red cell lysis, cells were counted and stained with a cocktail of antibodies for the detection of the indicated populations using anti-CD19-AF700, anti-CD23-PE/Cy7, anti-CD21-E450, anti-CD43-PE/Cy7, anti-GL7-FITC, anti-CD138-PE, anti-B220-E450, anti-B220-APC, anti-IL-10-APC, anti-CD3-FITC, anti-CD4-PerCP and anti-CD8-PE/Cy7 antibodies from eBioscience and Biolegend. Plasma cells were phenotyped as (Dump (CD3, CD4, CD8, CD11b, GR1 & CD11c)⁻ CD138⁺CD19⁻B220⁻) with the Dump⁺ markers identified using biotinylated antibodies and svPE. Alternatively, splenocytes were stimulated with 50 ng/ml PMA (Sigma-Aldrich, UK) plus 500 ng/ml ionomycin (Sigma-Aldrich, UK) and 10 μg/ml LPS (E. coli O111:B4, Sigma-Aldrich, UK) for 1 h before addition of 10 μg/ml Brefeldin A (Sigma-Aldrich, UK) and cells were then incubated for a further 5 h at 37 °C with 5% CO₂. Cells were fixed and washed several times in permeabilization buffer before anti-IL-10 was added in permeabilization buffer and incubated at 4 °C for 30 minutes. Cells were then washed three times with permeabilization buffer and finally with FACs staining buffer before data were acquired using a BD LSR II flow cytometer and analysis undertaken by FlowJo software (TreeStar).

PBMCs (3 × 10⁵) obtained by a Ficoll-Hypaque gradient were incubated 10 μg/mL of SEA for 16 h, 37°C, and 5% of CO₂. During the last 4 h of culture, Brefeldin A (10 μg/mL; Sigma, St. Louis, MO), which impairs protein secretion by the Golgi complex, was added to the cultures. Afterwards, the cells were stained with PERCPCy5.5-labeled antibody conjugated with CD14 (anti-CD14 PERCPCy5.5; clone 61D3), anti-CD16 FITC (clone CB16), anti-IL-10R PE (polyclonal), anti-IL-4Rα PE (clone hIL-4R-M57), and anti-HLA-DR PE (clone LN3) and then washed in PBS and fixed in 4% formaldehyde for 20 min at room temperature. Intracellular staining was performed with anti-IL-12 PE (clone C8.6) mAbs, anti-IL-10 APC (clone JES3-19F1), anti-TGF-β APC (clone 9016), anti-IL-6 APC (clone MQ2-13A5), and anti-TNF-α PECy7 (MAb11), all antibodies from eBioscience. The monocyte population was defined by nonspecific fluorescence from the forward scatter (FSC) and side scatter (SSC) as parameters such as cell size and granularity, respectively. Monocyte area corresponded to the specific region graph: region 1 (G1) (Figure 1(a)). A total of 100,000 events were acquired for all experiments.