Mice were sensitized by i.p. injection of 1 µg of mouse dinitrophenylated human serum albumin (DNP-HSA)–specific IgE (D8406; Sigma-Aldrich) in 100 µl PBS, and control mice were mock-injected i.p. with 100 µl of PBS. 16 h later, sensitized or non-sensitized control mice were injected i.p. with 250 ng of DNP-HSA (D8406; Sigma-Aldrich), and rectal temperature was measured every 10 min for a period of 60 min.
D8406
Manufactured by Merck Group
The D8406 is a piece of laboratory equipment designed for scientific analysis and research. It serves as a core function in various experimental setups without further interpretation on its intended use.
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Lab products found in correlation
5 protocols using d8406
1
Murine Allergic Response Assay
2
Murine Model of Allergic Skin Inflammation
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PCA was induced as follows82 (link): briefly, both ears of mice were intradermally injected with 10 ug/ml IgE (25ul, D8406, Sigma-Aldrich). The tail vein, 16 h later, was injected with 1 mg/ml DNP-HSA (100ul, D-5059, Biosearch Technologies). After 30 min, a full thickness biopsy of the right ear was fixed and embedded for toluidine blue staining. Evans blue was extracted from the remaining skin tissue of the right ear at 65 °C for 16 h, and its absorbance was measured at 610 nm. After 12 h, blood was collected, and the concentration of IL4, IL13, and IL10 in plasma/ear tissue was determined by ELISA according to the manufacturer’s instructions (Jianglai Biotechnology Co., Ltd., China). The concentration of LPS in plasma and histamine in plasma/ear tissue was determined by ELISA (USCN Life Science and Technology, China). The left ear tissue was frozen in liquid nitrogen and stored at −80 °C for subsequent qPCR detection.
3
Measurement of Mast Cell Degranulation
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Degranulation was detected by measuring β-hexosaminidase release (Lee et al., 2016 (link)). Briefly, RBL-2H3 cells (1×105 cells/well in a 24-well plate) were sensitized with 0.2 g/mL monoclonal anti-dinitrophenyl mouse immunoglobulin E (DNP-IgE, D8406, Sigma-Aldrich) overnight at 37°C in a 5% CO2 incubator. Then, the cells were washed twice with piperazine-N,N′-bis-(2-ethanesulfonic acid) (PIPES) buffer (pH 7.2) containing 25 mM PIPES, 110 mM sodium chloride (NaCl), 5 mM potassium (KCl), 5.6 mM glucose, 0.4 mM magnesium chloride (MgCl2), 0.1% bovine serum albumin (BSA), and 1 mM calcium chloride (CaCl2) to remove the DNP-IgE. After incubation with different concentrations of PD1 at 37°C for 30 min, the cells were treated with 1 μg/mL human DNP-albumin (DNP-hAb, A6661, Sigma-Aldrich), and then incubated for an additional 30 min at 37°C to induce degranulation. Then, 25 μL of the supernatant was transferred to a 96-well microplate and incubated for 2 h with 25 μL 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide (N9376, Sigma-Aldrich) in 0.1 M citrate buffer (pH 4.5). The reaction was terminated by adding 200 μL stop buffer (0.05 M sodium carbonate [Na2CO3/0.05 M sodium bicarbonate [NaHCO3], pH 10), and the optical density at 405 nm was measured.
4
Mast Cell Degranulation Assay Protocol
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Mast cell activation was assessed by β-hexosaminidase release in BMMCs84 (link),85 (link). In short, BMMCs were inoculated into 96 well plates with 8*103 cells per well and sensitized with 0.1ug/ml IgE (D8406, Sigma-Aldrich) prepared with serum-free medium. After sensitization for 24 h, the culture medium was discarded and LPS (L2630, Sigma-Aldrich) complete culture medium was added. After 6 h, the medium was discarded and 1 ug/ml DNP-HSA (D-5059, Biosearch Technologies) was added for 30 min.
The supernatant of each well was collected, and NP40 Lysis Buffer was added to each well to lyse the cells for 5 min. Lysate (50 ul) and supernatant (50 ul) were each incubated with substrate solution (50 μL) (3 mM 4-Nitrophenyl N-acetyl-β-D-glucosaminide, N9376, Sigma-Aldrich) for 90 min at 37 °C, then 150 uL of Na2CO3/NaHCO3 (pH = 10.6) were added to terminate the reaction, and the absorbance value was read at 405 nm by Cytation 5 multifunctional enzyme marker (BioTek, USA), and the degranulation ratio of mast cells was obtained by supernatant/(supernatant + lysate) absorbance value.
The supernatant of each well was collected, and NP40 Lysis Buffer was added to each well to lyse the cells for 5 min. Lysate (50 ul) and supernatant (50 ul) were each incubated with substrate solution (50 μL) (3 mM 4-Nitrophenyl N-acetyl-β-D-glucosaminide, N9376, Sigma-Aldrich) for 90 min at 37 °C, then 150 uL of Na2CO3/NaHCO3 (pH = 10.6) were added to terminate the reaction, and the absorbance value was read at 405 nm by Cytation 5 multifunctional enzyme marker (BioTek, USA), and the degranulation ratio of mast cells was obtained by supernatant/(supernatant + lysate) absorbance value.
5
Oxidative Stress and Proteasome Function
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