On day 0, male CD-IGS rats (170 – 190g; Charles River Laboratories) were anesthetized with isofluorane, their dorsal region shaved and 50 μl Anti-DNP-IgE (Clone SPE-7, Sigma, St. Louis, MO) that had been diluted 1:1000 in saline was injected via the intradermal route at 3 individual spots along the right side of the back. Saline was injected via the intradermal route at 3 individual spots along the left side of the back as a sham control. Twenty-four hours later, 200 μl of BSA-DNP (Calbiochem, San Diego, CA), prepared (1 mg prepared in a 2% solution of Evans blue dye: Sigma Chemicals, St. Louis, MO) was injected via the IV route via the tail vein. Thirty minutes after the BSA-DNP challenge the experiment was terminated and skin samples from the middle of each injection site taken for Evans blue dye measurement at an OD measurement at 620nm on a plate reader following incubation (2h at 90°C) and homogenization in formamide (Sigma Chemicals, St. Louis, MO). Vehicle (20% Captisol® (Captisol), PO), the positive control (Cromolyn: Sigma Chemicals, St. Louis, MO 100mg/kg, 2mL/kg, IV) or CC0484509 (3 and 10mg/kg, PO) were dosed 1h prior to the IV challenge with BSA-DNP.
Dnp bsa
Manufactured by Merck Group
Sourced in United States
DNP-BSA is a chemical compound used in various laboratory applications. It is a conjugate of 2,4-dinitrophenyl (DNP) and bovine serum albumin (BSA). The primary function of DNP-BSA is to serve as a hapten in immunological assays, allowing for the detection and quantification of analytes or target molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Formamide, by Merck Group (3 mentions)
Evans blue dye, by Merck Group (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Dnp ige, by Merck Group (2 mentions)
Dnp specific ige, by Merck Group (2 mentions)
Ova albumin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Histamine dihydrochloride, by Fujifilm (1 mentions)
Phenol red, by Fujifilm (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Tb green premix dimereraser, by Takara Bio (1 mentions)
Phosphate buffered saline (pbs), by Fujifilm (1 mentions)
Gum arabic, by Fujifilm (1 mentions)
Evans blue, by Fujifilm (1 mentions)
Dulbecco s modified eagle medium, by Fujifilm (1 mentions)
Hptlc plate, by Merck Group (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
22 protocols using dnp bsa
1
Rat Anaphylaxis Model for Compound Evaluation
2
Mast Cell Degranulation Assay Protocol
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Dulbecco’s modified Eagle medium (DMEM), 0.25 w/v% trypsin 1 mmol/L/EDTA 4Na solution with phenol red (trypsin/EDTA aqueous solution), phosphate-buffered saline (PBS), skim milk, DP, DX, histamine dihydrochloride, gum Arabic, Evans blue, and pontamine sky blue were obtained from FUJIFILM Wako Pure Chemical Co. Ltd. (Osaka, Japan). The RNeasy® Mini Kit was purchased from QIAGEN (Hilden, Germany). PrimeScript™ Reverse Transcriptase and TB Green® Premix Dimer Eraser™ were purchased from Takara Bio Inc. (Kusatsu, Japan). The dNTP mixture, random primer, and PolarScreen™ Glucocorticoid Receptor Competitor Assay Kit, Red, were purchased from Invitrogen (Waltham, MA, USA). HPTLC plate and DNP-BSA were purchased from Merck Millipore (Darmstadt, Germany). Fetal bovine serum (FBS) was purchased from Biosera (Boussens, France). The ceramide standards of ceramide (NS, NDS) and (AS) were purchased from Matreya LLC. (Philadelphia, PA, USA). Anti-DNP IgE was obtained from Seikagaku Industry (Tokyo, Japan).
3
Measuring RBL-2H3 Cell Degranulation
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RBL-2H3 cells (rat basophilic leukemia) were obtained from the Korean Cell Line Bank (KCLB, Korea) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. RBL-2H3 cells were seeded in 96-well plates (4 × 104 cells/well) and washed with DMEM containing 10% FBS after 24 h. After 48 h, cells were washed with DMEM containing 10% FBS and treated with 0.1 μg/ml anti-dinitrophenyl (DNP) IgE (Sigma). After 20 h, IgE-sensitized cells were washed twice with Tyrode buffer (pH 7.7) and incubated with SYK inhibitors. After 30 mins, cells were treated with Tyrode buffer containing 0.1 μg/ml DNP-BSA (Merck) for 1 h. The released β-hexosaminidase in supernatants was measured by addition of p-nitrophenyl N-acetyl-β-D-glucosaminide at 405 nm after 1 h.
4
Modulation of BMMC Proliferation and Cytokine Production
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One million BMMCs per mL were cultured in triplicate with IL-3 and SCF or 2 μg/mL DNP-IgE (Sigma Aldrich). To determine the effects of TSA on proliferation and cytokine production TSA in dimethyl sulfoxide (DMSO) was added in concentrations of 10, 30,100, 300, or 500 nM for varying time points. Control wells were treated with vehicle alone. Cells were then stimulated with 200 ng/mL DNP-BSA (Sigma Aldrich) or 20 ng/mL rIL-33 (Biolegend).
5
Cell Proliferation and IgE/PTX3 ELISA
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DMEM and fetal bovine serum (FBS) were purchased from Gibco. ELISA kits for IgE and PTX3 were purchased from Bethyl Laboratories, Inc. and R&D Systems, Inc., respectively. p-nitrophenyl-N-acetyl-β-D-glucosaminide (p-NAG), anti-DNP-IgE antibody, DNP-BSA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and o-phthalaldehyde were obtained from Sigma-Aldrich Co.
6
Mast Cell Degranulation Assay
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RBL-2H3 cells (2 × 105 cells/well) were sensitized with 200 ng/mL of dinitrophenyl- (DNP-) specific IgE (Sigma-Aldrich, St. Louis, MO, USA) overnight. After washing with PIPES buffer (25 mM PIPES at pH 7.2, 119 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgCl2·6H2O, 40 mM NaOH, 5.6 mM glucose, and 0.1% BSA), cells were treated with PBR fractions (25 and 50 μg/mL) or PP2 (Calbiochem, La Jolla, CA, USA), an Src tyrosine kinase inhibitor [1 (link), 39 (link)]. PP2 blocks the phosphorylation of Syk [1 (link), 39 (link)]. After 30 min, cells were stimulated with 200 ng/mL of antigen (DNP-BSA; Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37°C. Thereafter, the collected supernatant was mixed with 30 μL of 1 mM p-nitrophenyl-acetyl-β-D-glucosaminide (p-NAG; Sigma-Aldrich) in 0.1 M citrate buffer (0.1 M sodium citrate, 0.1 M citric acid, pH 4.5) and incubated at 37°C for 2 h. Then, 200 μL of 0.1 M Na2CO3/NaHCO3 solution (pH 10.0) was added to stop the reaction. Degranulation was calculated with the measurement of the released β-hexosaminidase as previously described [30 (link), 40 (link)].
7
Evaluating β-Hexosaminidase Release in RBL-2H3 Cells
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The β-hexosaminidase assay was performed as per the previous study [15 (link)]. RBL-2H3 cells were treated with 200 ng/ml of dinitrophenyl (DNP) specific IgE (Sigma-Aldrich, St. Louis, MO, USA). After being incubated overnight, cells were washed with the PIPES buffer (25 mM PIPES at pH 7.2, 119 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.4 mM MgCl2 6H2O, 40 mM NaOH, 5.6 mM glucose, and 0.1% bovine serum albumin (BSA)). GRC, GRC-SC11, or PP2 (Calbiochem, La Jolla, CA, USA) were treated for 30 min. Cells were stimulated with DNP-BSA (200 ng/ml, antigen; Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C. The absorbance was measured at 405 nm by a microplate reader (Epoch BioTek Instruments, Winooski, VT, USA).
8
MRGPRX2-Mediated Mast Cell Activation
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All cell culture reagents and DNP-specific mouse IgE (SPE-7) were purchased from Invitrogen (Carlsbad, CA, USA). Recombinant murine interleukin-3 (IL-3), stem cell factor (SCF), and recombinant human SCF were purchased from Peprotech (Rocky Hill, NJ, USA). DNP-BSA and p-nitrophenyl-N-acetyl-β-D -glucosamine (PNAG) were from Sigma-Aldrich (St. Louis, MO, USA), Compound 48/80 was from AnaSpec (Fremont, CA, USA). Amaxa transfection kit (Kit V) was purchased from Lonza (Gaithersburg, MD, USA). PE anti-human MRGPRX2 antibody was purchased from Biolegend (San Diego, CA, USA). Polyclonal MRGPRX2 Ab was purchased from Novus Biologicals (Littleton, CO, USA). HRP-conjugated anti-rabbit IgG was from Cell Signaling Technologies (Danvers, MA, USA). West Pico Chemiluminescent Substrate was from Thermo Scientific (Rockford, IL, USA). DNeasy Blood and Tissue Kit was purchased from Qiagen (Germantown, MD, USA). QuikChange II Site-Directed Mutagenesis Kit was purchased from Agilent Genomics (Santa Clara, CA, USA). Plasmid encoding hemagglutinin (HA)-tagged human MRGPRX2 in pReceiver-MO6 vector was obtained from GeneCopoeia (Rockville, MD, USA). Antimicrobial peptidomimetics (compound 1, compound 2, compound 3, compound 4, and compound 5) were obtained from Fox Chase Chemical Diversity Center (Doylestown, PA, USA).
9
Mast Cell Degranulation Assay
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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.
10
Antibody-Mediated Cellular Signaling
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Monoclonal anti‐dinitrophenyl (DNP) antibody, DNP‐BSA, and A23187 were obtained from Sigma (MO). Thapsigargin was obtained from Wako Pure Chemical Industries, Ltd. (Japan).
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