Anti cd4 mab

PECs or cultured cells were washed in FACS buffer (plus 2% FCS), followed by incubation with anti-CD16/CD32 (eBioscience, San Diego, CA, USA) for Fc blocking. We stained cells with anti-CD8, anti-CD5, anti-CD19, anti-B220, anti-F4/80 mAbs, anti-CD11b, and anti-CD11c (clone HL3) labeled with PE, FITC, or allophycocyanin (BD Biosciences, Chicago, IL, USA), or with FITC-labeled anti-Ly6G (clone 1A8), allophycocyanin-labeled anti-Ly6C (clone HK1.4), or anti-CD4 mAbs (eBioscience), or with Alexa Fluor 488-labeled anti-MGL1 (CD301a) mAb (AbD Serotec, Kidlington, UK) or control rat IgG2a mAb (R&D Systems, Minneapolis, MN, USA). For intracellular staining, we washed, permeabilized, and stained cells with PE-labeled anti-IL-12p35 or control murine IgG1 mAb (R&D Systems). For IL-12p35 (M1) and MGL1 (M2) subsets, gates were based on the exclusion of background staining with isotype control mAbs. We then washed, fixed, and acquired cells with the CellQuest software on a FACSCalibur system (BD Biosciences). For apoptosis detection, cells were first stained with allophycocyanin anti-CD8, washed, and then treated with FITC-annexin V (BD Biosciences) for 20 min at room temperature, according to the manufacturer; 7-AAD (eBioscience) was added just before flow cytometry. For analysis, we used the FlowJow software (TreeStar, Ashland, OR, USA).

For surface markers, MSCs were collected to stain with relevant fluorochrome-conjugated monoclonal antibodies (mAbs): anti-mouse CD29, CD90, CD44, CD34, CD45, and CD11b from eBioscience. Lymph nodes were isolated from mice and ground in culture medium. The suspensions were filtered through 70-μm cell strainers. For intracellular cytokine staining, single cell suspensions were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 μg/ml), and monensin (Enzo, 2 μg/ml). After 5 h, cells were stained with anti-CD4 mAb (eBioscience), fixed, permeabilized, and stained with anti-IFN-γ or IL-17 mAbs (eBioscience) according to the Intracellular Staining Kit (Invitrogen, Carlsbad, CA) instructions. For Treg cells staining, anti-CD4, anti-CD25, and anti-Foxp3 mAbs (eBioscience) were performed following Foxp3 Staining Buffer Set (eBioscience) protocols. Flow cytometry was performed using the BD FACSCanto II (Becton Dickinson) and data were analyzed using FlowJo software (Becton Dickinson) (24 (link)).

Icariin was purchased from MEYER company (Shanghai, China), whereas the antibodies used for western blotting were purchased from Cell Signaling Technology (Danvers, MA). The antibodies were anti-CD3 mAb (PE), anti-CD4 mAb (APC), anti-CD8a mAb (PE/Cy7), anti-CD25 mAb (APC/Cy7), anti-CD44 mAb (FITC), anti-CD62L mAb (APC/Cy7), anti-IFN-γ mAb (PE), HO-1 mAb (PE/Cy7), anti-CD16/32 mAb and anti-Foxp3 mAb (FITC) (eBioscience, San Jose, CA, USA).

CD4⁺ T cells (1 × 10⁶/well) isolated form spleen of control and SS model mice, or T cells (2 × 10⁵/well) isolated form salivary gland tissues of SS model mice were stimulated with Dynabeads mouse T-activator CD3/CD28 (Invitrogen) at bead/cell ratio of 1:4 and CCL22 (200 ng/mL) for 48 h, and were then cultured with phorbol myristate acetate (PMA; 50 ng/mL, Sigma-Aldrich, St. Louis, MO) and ionomycin (IM; 1 μg/mL, Sigma-Aldrich) in the presence of Brefeldin A (eBioscience) for the last 6 h. After washing, cells were stained with an anti-CD4 mAb, fixed in fixation/permeabilization solution (eBioscience); permeabilized in permeabilization buffer (eBioscience); and stained with anti-IFN-γ (eBioscience, XMC1.2), IL-4 (TONBO, 11B11), and IL-17A (eBioscience, TC11-18H10.1).

T lymphocyte cell proliferation was assessed by mixed lymphocyte reaction (MLR) cultures. Splenocytes were isolated from the cell suspensions by gradient centrifugation at 400 g for 25 min at 4°C with Lymphoprep (TBD Science, LTS1092PK). CD4⁺T cells (4 × 10⁶/ml) were labeled with 5 μmol/l Cell Proliferation eFlour 670 (eBioscience, USA) and then added into a 24-well plate. Allogeneic DC, DC-mock, and DC-IL10 had been pretreated with mitomycin C (Dakewe, Beijing, 25 μg/ml) for 30 minutes and then cocultured with CD4⁺T cells as responders (3 × 10⁵/well) from C57BL/6 mice at ratios of 1 : 5, 1 : 10, and 1 : 30 for 5 days. T cell proliferation reaction was examined by flow cytometric measurement with anti-CD4 mAb (eBioscience, USA).