RBL-2H3 Cell Culture, Stimulation and Treatment

Rat basophilic leukemia −2H3 (RBL-2H3) cell line was purchased from National Infrastructure of Cell Line Resource (Beijing, China) and cultured in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO₂ incubator. Cells were seeded at 4×10⁴ cells per well onto 96-well plates for TNF-α determination, 8×10⁴ cells per well on 48-well plates for β-hexosaminidase (β-Hex), toluidine blue and immunofluorescence detection, and 2×10⁶ cells in culture dishes for Western blotting (WB) analysis. The plated cells were sensitized with 50 ng/mL anti-DNP IgE (Sigma-aldrich) for 12 h, before being pretreated with PLE (50 μg/mL), RosS (5 μM), Vic-2 (5 μM), RosA (5 μM) and Mix (consisted of 2.535 μM RosS, 2.361 μM Vic-2 and 0.104 μM RosA, with a molar ratio of 50.70%: 47.22%: 2.08%), or with Mix, Syk inhibitor (BAY61-3606, 0.05 μM), PKC inhibitor (Rottlerin, 1 μM), NF-κB inhibitor (BAY11-7082, 2 μM) and cPLA₂ inhibitor (Arachidonyl trifluoromethyl ketone, ATK, 1 μM) for 1 h, and control cells were cultured under normal conditions with vehicle. Then, the cells were stimulated with 25 ng/mL DNP-BSA (Biosearch, California, USA) in PIPES buffer for 45 min, cell culture supernatants were collected for β-Hex detection and cells were used for toluidine blue staining. Or the cells were stimulated with 25 ng/mL DNP-BSA in complete DMEM medium for 12 h (cell culture supernatants were collected for TNF-α test) or 4 h (cells were used for immunofluorescence staining and WB analysis).