Bone marrow cells were harvested from the femur and tibiae of D01

Bone marrow cells were harvested from the femur and tibiae of D011.10 mice. Subsequently, the erythrocytes were lysed. After washing with 1% FCS supplemented RPMI 1640 medium, T and B cells were depleted using mouse pans T and B dynabeads (Invitrogen). T- and B-depleted cells were incubated at 37°C. After 4 h, nonadherent cells were harvested and cultured at 5 × 106 /mL in 24-well plate in complete medium (RPMI 1640 supplemented with 8% FCS, 2 mM L-glutamin, 5 × 10−5 M β-mercaptoethanol, streptomycin, nonessential amino www.selleckchem.com/products/ABT-263.html acids (Gebco) and 1 mM sodium pyruvate (Sigma-Aldrich)) with 1000 IU/mL of

rmGM-CSF (R&D systems), and 1000 IU/mL of rmIL-4 (R&D systems). The medium was refreshed every Selleckchem KU-60019 other day for 1 week. After 1 week culturing, bone marrow-derived DCs were harvested and cultured with DX5+CD4+, DX5−CD4+ T cells or their supernatants or medium for 3 days. LPS (0.01 μg/mL; Sigma-Aldrich) was added after 1 day. The DCs obtained were cultured at 0.4 × 106 /mL with OVA323-339 peptide and OVA-specific CD4+ T cells at 1 × 106 /mL in total volume of 150 μL for 3 days. After 3 days, cytokine production was determined by flow cyto-metry. IL-12

(20 ng/mL) that was added to the co-cultures of CD4+ T cells and DCs were purchased from eBioscience. The concentrations of anti-IL-4 and anti-IL-10 antibodies used for blocking studies were chosen on the Cell Penetrating Peptide basis of titration experiments where known concentrations of cytokine were effectively inhibited in a bioassay [45]. Cytokine levels in DCs cell culture supernatants were measured by ELISA using IL-12p70 kit ELISA Ready-set-Go (eBioscience) according to the manufacturer’s instructions. Matched pairs of antibodies to measure IL-12p40 were purchased from BD. The expression of the surface molecules was examined

using fluorescence-labeled antibodies against B7-H1 (MIH5) and B7-DC (TY25) from eBioscience and CD80 (16-10A-1), CD86 (GL-1), CD40 (3/23), and MHC class II from BD. CD4+ T cells were visualized by staining with anti-CD4-PerCP-Cy5.5 (L3T4/RM4-5; BD Pharmingen). KJ1-26-PE (Invitrogen) was used to detect OVA-specific T cells. Anti-IFN-γ-FITC (XMG1.2; BD Pharmingen) was used to detect IFN-γ-producing cells. The staining reactions were performed according to manufacturer’s protocol. In brief, the cells were first washed in the staining buffer (PBS containing 0.5% BSA); subsequently, the cells were incubated with antibodies for surface markers for 20 min at 4°C. For intracellular cytokine staining, Brefeldin A (10 μg/mL; Sigma-Aldrich) was added to co-culture of CD4+ T cells and DCs for 4 h. After washing, the cells were fixed using Cytofix/Cytoperm (BD Bioscience) followed by washing with Perm/wash (BD Bioscience). For determination of cytokine production, the cells were stained for intracellular cytokines in Perm/wash for 20 min.

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