Anti dnp ige

Anti-DNP IgE, DNP-bovine serum albumin (BSA), dexamethasone, and Evans blue were purchased from Sigma (St. Louis, MO, USA). The RBL-2H3 cell line (KCLB-22256) was purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin-streptomycin (PS) were purchased from Hyclone (Logan, UT, USA). The histamine, TNF-α, IL-6, IL-1β, and IL-4 ELISA kits were purchased from Biovision (Milpitas, CA, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-ERK (L352), anti-phospho-JNK (T183/Y185), anti-p38, anti-ERK, anti-JNK, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Boehmeria nivea is an eco-friendly pesticide-free product, which was purchased from a farming association (Yeonggwang, Korea).

5 × 10⁵ primary human mast cells were seeded overnight in the presence of 1 µg/ml anti-DNP IgE (Sigma Aldrich). The next day, half of the media was carefully removed and cells were stimulated for 5 h with the ligands indicated in each figure at a final concentration of 10⁶ cells/ml before RNA isolation. Cells were then harvested, centrifuged at 350 g for 5 min and RNA was isolated by column separation using the RNeasy Mini Kit (Quiagen) following manufacturer recommendations.

BMMC’s cultured for 4–6 weeks and seeded at 5 × 10⁴ cells per well in HEPES buffer were analyzed for β-hexosaminidase enzyme release following exposure of either ENMs or dinitrophenyl (DNP), as previously described40 (link)59 (link). Briefly, BMMC’s were sensitized overnight with anti-DNP IgE (Sigma- Aldrich, St. Louis MI) and treated with dinitrophenylated human serum albumin (DNP-HSA) (Sigma-Aldrich) for 30 min (100 ng/ml) to generate a positive control. Separately, non-sensitized BMMCs were exposed to ENMs at 25, 50, or 100 μg/ml concentrations (diluted to concentrations in diH₂O) with or without the addition of a bovine serum albumin (BSA) biocorona for 1 h. Following incubation at 37 °C, supernatant and lysed cells were treated with p-nitrophenyl-N-acetyl-β-d-glucopyranoside, a substrate for N-acetyl-β-D-hexosaminidase (β-hexosaminidase), for 90 min and analyzed at 405 nm using an uv/vis spectrometer (BioTeck Instruments Inc., Winooski, VT). β-hexosaminidase release was calculated as percent total cell content after subtracting background release from untreated control groups and ENM-only control groups to ensure there was no interference with the biochemical assay. All experiments were performed in triplicate from 3 individual batches of mature mast cells grown in IL-3 supplemented media (n = 3/group).

RBL-2H3 cells were cultured in RPMI 1640 (Nacalai Tesque, Tokyo, Japan) supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 IU/ml), and streptomycin (100 IU/ml) at 37°C in a humidified 5% CO₂ atmosphere. Cells were sensitized with anti-DNP IgE (1 μg/ml) and stimulated with DNP-HSA (50 ng/ml; Sigma-Aldrich, St Louis, MO, USA). The β-hexosaminidase release assay was performed according to an established method (16 (link)). Briefly, the collected supernatant was incubated with p-nitrophenyl-N-acetyl-β-D-glucosaminide (2 mM) dissolved in 0.1 M citrate buffer (pH 4.5) for 1 h at 37°C. 0.1 M carbonate buffer (pH 10) was then added to the reaction wells to stop the reaction and develop color. Colorimetric measurements were performed at a wavelength of 405 nm using the Tecan Infinite F50 microplate reader (Tecan, Mannedorf, Switzerland).