The expression levels of cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-8) in the culture supernatants were measured using an enzyme-linked immunosorbent assay. In this experiment, 96-well microtiter plates were coated with capture TNF-α and IL-8 antibodies (Invitrogen, Carlsbad, CA, USA) and then incubated overnight at 4 °C. The plates were blocked with phosphate-buffered saline (PBS) containing 10% skimmed milk for 1.5 h and then washed three times with PBS containing 0.05% Tween 20. After the culture supernatants were added, the plates were incubated for 1 h and washed again three times. They were added to TNF-α and IL-8 antibodies separately, incubated for 1 h, and then washed again three times. Subsequently, they were incubated with 400-fold diluted streptavidin (Proteintech Group, Inc., Rosemont, IL, USA) at room temperature for 30 min. Finally, a coloring reagent, 4 mg/mL O-phenylenediamine (Wako Pure Chemical Industries, Ltd., Osaka, Japan), was prepared in citrate buffer with 0.01% H2O2 and then added to the wells. After 15 min, the reaction was stopped using 6 N sulfuric acid. Absorbance was determined at 492 nm using a MULTISKAN FC microplate reader (Thermo Fisher Scientific K.K. Tokyo, Japan).
O phenylenediamine
Manufactured by Fujifilm
Sourced in Japan
O-phenylenediamine is a chemical compound used in various laboratory applications. It serves as a precursor for the synthesis of dyes, pharmaceuticals, and other organic compounds. The core function of O-phenylenediamine is to provide a versatile building block for the development of diverse chemical products.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc microplate reader, by Thermo Fisher Scientific (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Hydrogen peroxide, by Fujifilm (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Trichloroacetic acid, by Fujifilm (1 mentions)
Protein a sepharose fast flow column, by GE Healthcare (1 mentions)
Dnase 1, by Nacalai Tesque (1 mentions)
Mouse monoclonal anti tubulin antibody, by Merck Group (1 mentions)
Rnase a, by Nacalai Tesque (1 mentions)
Proteinase k, by Merck Group (1 mentions)
T1503, by Merck Group (1 mentions)
H2so4, by Nacalai Tesque (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
4 protocols using o phenylenediamine
1
Cytokine Expression Quantification Assay
2
Quantifying H. pylori Antibodies
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The levels of serum IgG subclass and total IgG antibodies against H. pylori antigen (cagA; ABD, vacA; s1c/m1, cagE+, iceA1, and babA2) were measured by ELISA. Ninety-six-well microtiter plates were coated with H. pylori lysate (50 μg/ml) in 100 μl of 0.1 M carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. After the wells were blocked with Phosphate Buffered Saline (PBS) containing 10% skim milk, the plates were incubated with sera containing 1:1000 of IgG and 1:100 of IgG1/2 for 2 h and washed with PBS containing 0.05% Tween 20. Peroxidase-labeled rabbit anti-human IgG1, IgG2, or IgG antibody (Dako Japan) was then added subsequently, and the plates were incubated for 2 h. After the plates were washed, 1 mg/ml of o-phenylenediamine (Wako Pure Chemical) in citrate buffer (pH 5.5) was added to the wells. The ODs were measured at 490 nm using an ELISA plate reader (Bio-Rad).
3
Radioimmunoprecipitation Assay (RIPA) Protocol
4
AMIGO2 Monoclonal Antibody Production and Detection
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Hybridoma cells producing AMIGO2-specific mAb were identified using ELISA. In brief, a 96-well immunoassay plate (44-2404, Thermo Fisher Scientific) were coated with 50 µg/well antigen, washed three times with PBS-T (0.05% v/v Tween-20: 160-21211, Fujifilm Wako Pure Chemical), and blocked with PBS containing 5% skim milk (232100, Difco, Detroit, MI) for 30 min. After incubation with 100 µL of serially diluted serum samples or supernatant for 1 h, plates were again washed and incubated with 100 µL of anti-rat IgG (H+L)-HRP conjugate (A110-105P, Bethyl Laboratories, Montgomery, TX) diluted 50,000-fold in TBS-T (50 mM Tris-HCl, pH 8.0) (T1503, Sigma-Aldrich), 150 mM NaCl (195-01663, Fujifilm Wako Pure Chemical), 0.05% v/v Tween 20) for 30 min. Plates were washed again as described above, developed using 100 µL of o-phenylenediamine (160-11022, Fujifilm Wako Pure Chemical) for 30 min, and stopped using 25 µL of 1 M H2SO4 (95626-06, Nacalai Tesque, Kyoto, Japan). Absorbance was measured at 492 nm by microplate reader Epoch2 (Bio Tek, Winooski, VT).
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