Mast cells were adjusted to 1×10 5 cells/mL in RPMI 10 and incubated in the presence or absence of PEG (10 g/mL) and josamycin (10 M;
provided by Astellas Pharma Inc., Tokyo, Japan) for 18h at 37C in a humidified atmosphere with 5% CO2, and then washed before use. Next, T helper (Th) cells were separated from DO 11.10 TCR Tg mouse spleen cells using an EasySep Negative Selection Mouse CD4 + T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and then treated with mouse anti-CD62L monoclonal antibody (clone lam1-116, IgG2a) (1 g per 1 × 10 6 cells; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 10 for 1 h on ice. The Th cells that had been reacted with anti-CD62L antibody were then purified using a CELLection TM Pan Mouse IgG Kit (Invitrogen Dynal AS, Oslo, Norway), and used as naïve Th cells. The naïve Th cells (5 × 10 5 cells/mL) were cultured with the above mast cells (1 × 10 5 cells/mL) in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 days at 37°C. The cells were then stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 500 ng/mL ionomycin (Sigma) for 24 h at 37°C. The cell supernatants were finally removed and tested for production of interferon (IFN)- and IL-4 using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).
provided by Astellas Pharma Inc., Tokyo, Japan) for 18h at 37C in a humidified atmosphere with 5% CO2, and then washed before use. Next, T helper (Th) cells were separated from DO 11.10 TCR Tg mouse spleen cells using an EasySep Negative Selection Mouse CD4 + T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and then treated with mouse anti-CD62L monoclonal antibody (clone lam1-116, IgG2a) (1 g per 1 × 10 6 cells; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 10 for 1 h on ice. The Th cells that had been reacted with anti-CD62L antibody were then purified using a CELLection TM Pan Mouse IgG Kit (Invitrogen Dynal AS, Oslo, Norway), and used as naïve Th cells. The naïve Th cells (5 × 10 5 cells/mL) were cultured with the above mast cells (1 × 10 5 cells/mL) in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 days at 37°C. The cells were then stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 500 ng/mL ionomycin (Sigma) for 24 h at 37°C. The cell supernatants were finally removed and tested for production of interferon (IFN)- and IL-4 using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).