Jes3 9d7

Whole blood was collected in Vacutainer tubes containing EDTA (Becton, Dickinson, USA) and 100μL aliquots were mixed in tubes with 2μL of undiluted monoclonal antibodies anti-CD14 (clone MΦP9) conjugated with peridinin chlorophyll protein complex (PerCP) (BD Pharmingen^™, USA). After erythrocyte lysis, cells were washed, permeabilized and incubated with monoclonal antibodies against MMP-2 (1A10) and MMP-9 (56129) (R&D Systems, USA) and IL-1β (AS10), TNF-α (6401.1111), TGF-β (9016) or IL-10 (JES3-9D7) (BD Pharmingen^™, USA) conjugated with phycoeritrin (PE) or fluorescein isothiocyanate (FITC). After incubation, cells were fixed, and phenotypic analyses were performed by flow cytometry using FACSCalibur cytometer (BD Biosciences, Breda, Netherlands). A total of 7x10⁴ neutrophils and monocytes (gated based on forward and side scatter plot) were collected and data were analyzed using FlowJo software. Neutrophils and monocytes analysis was performed using a dot plot of anti-CD14 (FL3)/SSC. The neutrophils were gated as SSC^HighCD14^low and monocytes were gated as SSC^lowCD14^High [30 (link)].

Cytokine levels in supernatants were measured by ELISA, using antibody pairs for the following cytokines: IL-1β (CT213-c; 1:400 and CD213-d; 1:400), IL-6 (CD205-c; 1:200 and CD205-d; 1:200), IL-13 (QS-13; 1:200 and LM-1; 1:100), IL-23 (CD517-c; 1:400 and C8.6; 1:4,000), IFN-γ (MD2; 1:500 and MD1; 1:500; all from U-CyTech), IL-10 (JES3-9D7; 1:1,000 and JES3-12G8; 1:1,000; BD Pharmingen), TNF-α (MAb1; 1:500 and MAb11; 1:250) and IL-17A (eBio64cap17; 1:1,000 and eBio64dec17; 1:1,000; both from eBioscience).

Expanded CD4⁺CD25⁺ cells were stimulated with phorbol myristate acetate (2 ng ml⁻¹) and ionomycin (500 ng ml⁻¹) (Sigma) for 24 h. Monensin (GolgiStop, BD Biosciences) was added for the last 4 h of incubation. Cells were fixed and permeabilized using Human FOXP3 Buffer Set, followed by staining with fluorochrome-conjugated IL-10 (JES3-9D7) and IL-17A (N49-653-19F1, BD Biosciences) mAbs.