The effect of SHE on the β-hexosaminidase release from mast cells was evaluated according to the method reported by this research [37 (link)]. Briefly, BMCMCs (2 × 105/well) pre-incubated with various concentrations of SHE for 2 h were sensitized by anti-DNP-IgE for 1 h and stimulated by DNP-BSA (LSL Japan Inc., Tokyo, Japan, 100 ng/mL). After 1 h, the supernatant was obtained from the cells and the cells were lysed with Tyrode’s buffer containing 0.5% Triton X-100. Then, 0.1 M sodium citrate substrate buffer (pH 4.5) containing 4-p-nitrophenyl-N-acetyl-β-d- glucosaminide 1.3 mg/mL was added to the supernatants and the cell lysates. The mixtures were reacted for 1 h at 37 °C. The reaction was terminated by adding 0.2 M glycine (pH 10.7). The absorbance of each mixture was measured at 405 nm using a microplate reader. Histamine release was evaluated using a Histamine EIA kit (Oxford Biomedical Research, Oxford, MI, USA).
Histamine eia kit
Manufactured by Oxford Biomedical Research
Sourced in United States
The Histamine EIA kit is a quantitative in vitro diagnostic test used to measure the concentration of histamine in biological samples, such as plasma or serum. The kit utilizes an enzyme-linked immunosorbent assay (EIA) technique to detect and quantify the target analyte.
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6 protocols using histamine eia kit
1
Evaluating β-Hexosaminidase Release from Mast Cells
2
Measuring Histamine Levels in Plasma and Cells
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The amounts of histamine in the mouse plasma and in the culture supernatants were measured with histamine EIA kit (Oxford Biomedical research, Inc., Oxford, MI, USA).
For some experiments, the changes of histamine concentrations in the culture supernatants during the indicated culture duration (e.g. 4~8 hours, 8~12 hours, 12~16 hours, 16~20 hours, 20~24 hours) were calculated and expressed each of them as the amounts of net histamine release from mast cells during the culture duration. The plasma or culture supernatant samples were not taken from the same mice or cultures, but from the different mice or cultures except the experiments inFig. 4E .
For some experiments, the changes of histamine concentrations in the culture supernatants during the indicated culture duration (e.g. 4~8 hours, 8~12 hours, 12~16 hours, 16~20 hours, 20~24 hours) were calculated and expressed each of them as the amounts of net histamine release from mast cells during the culture duration. The plasma or culture supernatant samples were not taken from the same mice or cultures, but from the different mice or cultures except the experiments in
3
Histamine Quantification Assay
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The levels of histamine in culture supernatants or mouse plasma were measured with the histamine EIA kit (Oxford Biomedical Research, Oxford, MI, USA).
4
Peanut-specific IgE and IgG1 ELISA
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mMCPT-1 was detected using the MCPT-1 mouse uncoated ELISA kit (Thermo Fisher) following the manufacturer’s protocol. Histamine was assayed using Histamine EIA kit (Oxford Biomedical Research) according to the manufacturer’s protocol. For the peanut-specific IgE ELISA, sera from individual mice were added to peanut-coated Maxisorp immunoplates (Nalge Nunc). Peanut-specific IgE antibodies were detected with goat anti-mouse IgE-unlabelled (Southern Biotechnology) and rabbit anti-goat IgG-alkaline phosphatase (Invitrogen) and developed with p-nitrophenyl phosphate (SeraCare Life Sciences). For peanut-specific IgG1 ELISA, sera from individual mice were added to peanut-coated Maxisorp immunoplates (Nalge Nunc). Peanut-specific IgG1 was detected using goat anti-mouse IgG1-HRP conjugated (Southern Biotechnology Associates) and TMB liquid substrate system for ELISA (Sigma Aldrich). The plates were read in an ELISA plate reader at 405 nM (IgE) or 450 nm (IgG1). Optical density values were converted to nanograms per millilitre of IgE or IgG1 by comparison with standard curves of purified IgE or IgG1 using linear regression analysis and are expressed as the mean concentration for each group of mice ± s.e.m.
5
Quantifying Histamine in Cellular Samples
6
Measuring Inflammatory Markers in PSA
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Serum samples were collected from the mice at 10 minutes or 180 minutes after induction of PSA reactions. The amounts of MCP-1 (CCL2) and IL-6 in the serum at 180 minutes after induction of PSA reaction were measured using the mouse MCP-1 and IL-6 ELISA kits (R&D, Minneapolis, MN). The amounts of histamine in the serum at 10 minutes and 180 minutes after induction of PSA were measured using histamine EIA kit (Oxford Biomedical research, Inc., Oxford, MI, USA).
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