Dnp ige

Aliquots of BMDMCs were incubated overnight with anti-mouse monoclonal dinitrophenyl (DNP)-IgE (100 ng/ml; Sigma) to sensitize the cells, and the following day, the cells were activated by adding DNP-BSA (1 µg/ml; Sigma-Aldrich, Dorset, UK). Cell-free supernatants were collected at 1 h to measure histamine and/or PGD2 release. Aliquots were stored at -70°C for subsequent analysis. When drugs or antibodies were tested, these were added to cells 5 min prior to IgE cross-linking.

ICR mice (7 weeks old male) were intradermally injected with 80 ng of mouse anti-dinitrophenyl (DNP) IgE (Sigma) into one ear. After 24 h, spinacetin or dexamethasone (Dexa) was orally administered. One hour later, the mice were intravenously challenged with 60 μg of DNP-HSA in 200 μl of PBS containing 1% (w/v) Evans blue. The mice were euthanized 1 h after treatment with DNP-HSA. The ears were removed, and then dissolved with 400 μl of formamide at 63°C overnight to measure the amount of dye extravasated by Ag. The absorbance of dye was measured at 630 nm using a microplate reader (BIO-RAD iMark, Hercules, CA, United States). The ears were fixed with 4% formaldehyde and embedded in paraffin. Four micrometer sections of the tissues were prepared and stained with toluidine blue to count the number of mast cells. All experiments using animals were approved on Oct 27, 2017, by the Institutional Animal Care and Use Committee of Tianjin Medical University (No. of allowance: TMUaMEC2017022).

Mice were intradermally sensitized with 100 μg dinitrophenyl-specific IgE (DNP-IgE, Sigma) in one ear and PBS as the control in the other ear. After 16–18 hours, mice were retroorbitally challenged with 100 μg DNP-coupled albumin (DNP-HSA, Sigma). Ear thickness was measured at 0, 1, and 24 hours after challenge, as previously described [6 (link)]. Ears were harvested 24 hours after challenge. Cellular infiltration was analyzed by histology of tissue sections and cytospun cells as well as by flow cytometry. Cytokine expression was determined by ELISA; obtained values were normalized to total protein determined by Pierce BCA protein assay kit (Thermo Scientific). In some experiments, donor basophils were retroorbitally transferred into recipients 1 hour before challenge. Differences in ear thickness were determined by subtracting the baseline 0-hour measurement from the measurement at 1 or 24 hours after challenge.

RBL-2H3 cells were seeded into 24-well plates at 6 × 10⁴ cells/well and incubated for 24 h. The cells were then cultured for 1 h in serum-free medium containing vehicle (DMSO) or undecane (0.5, 1 and 5 μM), followed by sensitization with 50 ng/mL of DNP-IgE (Sigma Aldrich, St. Louis, MO, USA) for 4 h. The sensitized cells were washed two times with PBS and degranulation was induced by 50 ng/mL of DNP-HSA (Sigma Aldrich, St. Louis, MO, USA). After 1 h, the cells were washed again with cold PBS and fixed in 4% paraformaldehyde (Nest Biotechnology, Rahway, NJ, USA) at 25 °C. In the next step, 0.1% toluidine blue (Sigma Aldrich, St. Louis, MO, USA) in 1% sodium chloride (pH 2.5) was added to each well and cells were incubated for 30 min. After that, the stained cells were washed three times with PBS. Images of the stained cells were captured with an Olympus IX71 microscope using a DP-70 controller (Olympus, Center Valley, PA, USA).

The herbs of Saposhnikovia divaricata (whole root) was purchased in July 2020 from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China), and collected from Heilongjiang Province, China. The rat basilic leukaemia cell line 2H3 (RBL-2H3) was obtained from Chinese Academy of Sciences. Ketotifen Fumarate (KF) was obtained from Sigma-Aldrich (St. Louis, MO, USA). DNP-IgE was obtained from Sigma-Aldrich. DNP-BSA was obtained from Biosearch. Primers were synthesised by Sangon Biotech Co., Ltd. (Shanghai, China). POG standard (purity (HPLC) ≥ 98.0%) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Takara RNAiso Plus kit, reverse transcription agent cassette record and quantitative PCR kit were obtained from Takara.
Preparation of SD decoction: 2 g SD root standard was added 20 mL (10 times amount) of ultrapure water, soaked for 0.5 h, then refluxed in water bath at 100 °C for 1 h, and the filtrate was collected after suction filtration. 20 mL (10 times amount) of ultrapure water was added into filter residue, refluxed in water bath at 100 °C for 1 h, and the filtrates was collected together after suction filtration. A solution containing SD at a concentration of 50 mg/mL was obtained, and store at −20 °C after filtration with 0.22 μm microporous membrane.

RBL-2H3 cells (rat basophilic leukemia) were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The cell culture media, trypsin (0.25%), and antibiotics (penicillin and streptomycin) were obtained from Hyclone (Logan, UT, USA). Dimethyl sulfoxide (DMSO), lipoxygenase from glycine max (soybean), DNP-IgE, DNP-HSA, 4-nitrophenyl-N-acetyl-β-D-glucosaminide, 1-chloro-2,4-dinitrobenzene (DNCB), toluidine blue O, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium carboxymethylcellulose (CMC), and formaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Samchun Pure Chemical Co. (Gyeonggi-do, Korea). The cDNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All primers for checking the mRNA of markers were synthesized by Macrogen (Seoul, Korea). The qPCRBIO SyGreen Blue Mix Lo-ROX was obtained from PCR Biosystems, Inc. (Wayne, PA, USA). All antibodies used in this research were from Cell Signaling Technology (Beverly, MA, USA), except for β-actin, which was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).