Dnp bsa

Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), fetal bovine serum (FBS), and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Minimum Essential Medium Eagle, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lipopolysaccharide (LPS), 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG), and monoclonal anti-DNP-IgE were supplied by Sigma–Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Gaithersburg, MD, USA). Primary and secondary antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA), Cell signaling (Danvers, MA, USA), and Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The murine macrophage RAW 264.7 cell line was obtained from KCLB (Seoul, Republic of Korea). The rat basophilic leukemia RBL-2H3 cell line was obtained from ATCC (Manassas, VA, USA).

Saponarin (purity > 98%) was obtained from Extrasynthese (Genay, France). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biowest (Kansas City, MO, USA), and lipopolysaccharide (LPS), monoclonal anti-DNP-IgE, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Carlsbad, CA, USA). Recombinant human TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ, USA). Primary anti-bodies against ERK, JNK, p38, Lyn, Syk, PLCγ, STAT1, p-ERK, p-JNK, p-p38, p-STAT1, p-Lyn, p-Syk, p-PLCγ, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against TSLP was purchased from ABclonal (Woburn, MA, USA). Primary antibodies against FcεRIγ were purchased from LSBio (Seattle, WA, USA). The SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea). RAW264.7 (ATCC^® TIB-71) and RBL-2H3 (ATCC^® CRL-2256) cells were purchased from the American Type Culture Collection (ATCC). HaCaT cells were obtained from Byoung-Woo Lim of Konkuk University, Korea.

BMMC were sensitized with 500 ng/mL mouse anti-DNP IgE (SPE-7, Sigma) for 16 h or cultured in fresh media without IgE (unsensitized). The BMMC were washed with fresh media and placed onto the CNC/agarose composites (or standard untreated cell culture wells as controls) at a density of 15,000 cells/well and IgE sensitized cells were stimulated with 10 ng/mL DNP-BSA (Invitrogen) and unsensitized cells were stimulated with 1 μM A23187 for 18 h in a humidified atmosphere at 37°C with 5% CO₂. After treatment, the cells were spun at 200 rcf (× g) for 5 min, and 50 μL of the supernatants were collected and aliquoted into a fresh 96-well plate. For total LDH wells, 50 μL of 0.1% Triton (Fisher, Hampton, NH, United States) was added to lyse the cells. Fifty μL of the LDH assay reagent (iodonitrotetrazolium dinucleotide sodium salt (4 mM), β-nicotinamide adenine dinucleotide sodium salt (6.44 mM), lithium L-lactate (320 mM), and TrisBase [2-amino-2-(hydroxymethyl)-1,3-propanediol (pH 8.2, 0.2 M)] was added to the wells and left to incubate in the dark at room temperature for 90 min. To stop the reaction, 50 μL of acetic acid (1 M) was added to the wells, and the absorption was measured using a Varioskan Lux microplate reader (ThermoFisher) at 490 nm with a reference wavelength of 680 nm.

BMMC were seeded at a density of 1 × 10⁶ cells/mL onto the CNC/agarose composites or standard untreated cell culture plates as controls and incubated for 18 h. BMMC were then washed and sensitized with 500 ng/mL mouse anti-DNP IgE (SPE-7, Sigma) or cultured in fresh media without IgE (unsensitized control) for 18 h. BMMC were then stimulated with either 10 ng/mL DNP-BSA (Invitrogen) or 1 μM A23187 for 18 h at 37°C. Following activation, cell suspensions were centrifuged at 200 g × for 5 min and cell-free supernatants were collected. Supernatants were analysed via commercial ELISA (ThermoFisher) and Meso Scale multiplex assays (Meso Scale Discovery, Rockville, MD, United States) following manufacturer instructions, and plates were read using the Varioskan Lux (ThermoFisher) and MESO QuickPlex SQ 120 MM (Meso Scale Discovery) plate readers respectively.

MC/9 were seeded at a density of 1 × 10⁶ cells/mL in a 24-well plate and treated with resveratrol or quercetin for 1 h or 24 h in a humidified chamber at 5% CO₂ and 37 °C followed by sensitization with 0.5 µg/mL IgE (SPE-7, Sigma-Aldrich) for 18 h and stimulation with 100 ng/mL DNP-HA (Invitrogen) for 24 hr. Supernatants were collected and used to assess de novo synthesis of TNF. The ELISAs were performed according to manufacturer’s instruction (Applied Biosystems, Waltham, MA, USA) and data was quantified using a standard curve.
BMMC were seeded at a density of 1 × 10⁶ cells/mL in a 24-well plate, sensitized with 0.5 µg/mL IgE (SPE-7; Sigma-Aldrich) and treated with 10 µM of resveratrol or quercetin for 24 h in a humidified chamber at 5% CO₂ and 37 °C followed by stimulation with 10 ng/mL DNP-BSA (Invitrogen) for 24 h. Supernatants were collected and used to assess de novo synthesis of TNF. The ELISAs were performed according to manufacturer’s instruction (Applied Biosystems) and data was quantified using a standard curve.

Alcian Blue 8GX, safranin O, lipopolysaccharides (rough strains) from Salmonella enteric serotype Minnesota Re 595 (Re mutant), mouse IgE-anti-DNP, and JNK-IN-8 were all purchased from Sigma Aldrich (Saint Louis, MO, USA). Human myeloma IgE was obtained from Calbiochem-Merck (Darmstadt, Germany). Polyclonal rabbit anti-human IgE Ab was obtained from Dako (Copenhagen, Denmark). Goat anti-rat IgG F(ab)₂ Ab was from Jackson ImmunoResearch Laboratories (Baltimore, MD, USA). DNP-BSA and Rat myeloma IgE were purchased from Invitrogen (Carlsbad, CA, USA). STF-083010 was synthesized by Tocris Bioscience (Bristol, UK). Thapsigargin was synthesized by Fermentek (Jerusalem, Israel), while DMSO was from Bio-Lab (Jerusalem, Israel).

The degranulation response of RBL-2H3 cells was quantified by measuring the level of β-hexosaminidase released in the supernatants [20 (link)]. RBL-2H3 cells were grown on 6-well plates at a density of 2 × 10⁵ cells for 24 h. The cells were then treated with 800 ng/mL DNP-specific IgE (Sigma-Aldrich) overnight. To remove the excess IgE, the cells were washed twice with Tyrodes’ assay buffer (pH 7.3). The cells were then stimulated with DNP-BSA (Invitrogen), suspended in 500 µL of extracellular buffer with 0.1% BSA, and incubated at 37 °C for 1 h. After incubation, 50 µL of the supernatant was incubated with 200 µL of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich) in 0.05 M citrate buffer (pH 4.5) at 37 °C for 1 h. The enzyme reaction was terminated by adding 500 µL of sodium carbonate buffer. The OD of each reaction was recorded at 405 nm. The percentage of viable cells was calculated using the following equation (Equation (3)): $\beta -\mathrm{hexosaminidase} \sec \mathrm{retion} (\%)=\frac{Am}{\left(Al-Am\right)}\times 100$
where, Al is the absorbance of the cell lysate and Am is the absorbance of the medium.