Culture supernatants were collected after 72 h incubation of lymph node cells with LmAg, and analyzed for the production of IL-4, IL-10, IFN-γ and TNF-α cytokines using the Sandwich ELISA method. Briefly, 96 well plates were coated with Capture antibodies for overnight, blocked and culture supernatants or standards were added for overnight. Plates were washed, respective biotinylated secondary antibodies were added, streptavidin-AKP and PNPP (p-nitrophenyl phosphate) substrates were used for detecting secondary antibodies. Capture and detection antibodies were obtained from BioLegend, San Diego, CA and cytokine standards were obtained from BD biosciences, San Jose, CA. The cytokine production was quantified by measuring the absorbance at 405 nm using Spectramax microplate reader and Softmax Pro software (Molecular Devices LLC, Sunnyvale, CA, USA).
Cytokine standards
Manufactured by BD
Cytokine standards are reference materials used to calibrate and validate instruments and assays for the quantitative measurement of cytokines in biological samples. They provide a consistent and reliable means to ensure accuracy and reproducibility in cytokine detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Capture and detection antibodies, by BioLegend (1 mentions)
Spectramax microplate reader, by Molecular Devices (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
Human tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Human inflammatory cytokine kit, by BD (1 mentions)
Ultracel 30 membrane, by Merck Group (1 mentions)
2 protocols using cytokine standards
1
Cytokine Profiling of LmAg-Stimulated Lymph Cells
2
Quantifying Cytokine Levels in Cell Cultures
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To analyze excreted TNFα levels, KBM-7-shBRCA2 or KBM-7-shLUC cells were treated with doxycycline (1 μg per mL) for 48 h. Proteins in supernatant culture media were concentrated using Microcon-30 kDa centrifugal filter units with Ultracel-30 membrane (Millipore). Subsequently, TNFα concentrations were determined using a human TNFα ELISA kit (KHC3011, Life Technologies).
IL-6, IL-8 and IL-10 levels were analyzed using the Human Inflammatory Cytokine Kit (BD Bioscience, #551811), according to the manufacturer’s protocol. In short, media were collected from BT-549 cells harboring different shRNAs, after treatment with doxycycline for 0, 2 or 4 days. Media samples (50 μL per sample) were incubated with IL-6, IL-8 and IL-10 capture beads for 3 h at room temperature. After two wash steps, samples were measured on an LSR-II (Becton Dickinson). Data were analyzed using FlowJo software, and cytokine concentrations were calculated using cytokine standards (BD Bioscience).
IL-6, IL-8 and IL-10 levels were analyzed using the Human Inflammatory Cytokine Kit (BD Bioscience, #551811), according to the manufacturer’s protocol. In short, media were collected from BT-549 cells harboring different shRNAs, after treatment with doxycycline for 0, 2 or 4 days. Media samples (50 μL per sample) were incubated with IL-6, IL-8 and IL-10 capture beads for 3 h at room temperature. After two wash steps, samples were measured on an LSR-II (Becton Dickinson). Data were analyzed using FlowJo software, and cytokine concentrations were calculated using cytokine standards (BD Bioscience).
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