Cells were treated as indicated for 48 h, and supernatants were collected. Cells and cell debris were removed from samples by centrifugation at 5000× g for 5 min, and the samples were kept at −80 °C until used. Blood samples from mice were collected into tubes with EDTA from the orbital sinus, and then the blood cells were removed by centrifugation at 10,000× g for 10 min, and the plasmas were stored at −80 °C until used. Concentrations of IFN-γ, TNF-α, and IL-2 were determined using Human IFN-γ ELISA Kit II, Human TNF-α ELISA Kit II, and Human IL-2 ELISA Kit II (BD Biosciences, San Jose, CA, USA), respectively, according to the manufactures’ instructions.
Human ifn γ elisa kit 2
Manufactured by BD
Sourced in United States
The Human IFN-γ ELISA Kit II is a quantitative sandwich enzyme immunoassay designed for the measurement of human interferon-gamma (IFN-γ) in cell culture supernatants, serum, and plasma. The kit utilizes a polyclonal antibody specific for human IFN-γ coated on a microplate. Samples and standards are pipetted into the wells, and any IFN-γ present is bound by the immobilized antibody. After washing, an enzyme-linked polyclonal antibody specific for human IFN-γ is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added, and the color that develops is measured.
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4 protocols using human ifn γ elisa kit 2
1
Cytokine Quantification in Mouse Plasma
2
Quantifying Cytokine Responses in Macrophages Treated with ECM
3
T-cell Cytokine Production Assay
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CD4-positive cells were re-suspended in culture medium (RPMI-1640 containing inactivated FCS, 2-ME, 3% glutamine solution, penicillin, and streptomycin), and centrifuged at 120 × g for 15 minutes. Pellets were adjusted to 1 × 106 cells/mL with culture medium. CD4-positive cells were stimulated with concanavalin A, and incubated at 37°C for 24 hours. After centrifugation, the supernatant was removed; cytokine levels were determined using an enzyme-linked immunosor-bent assay (ELISA) kit. IFN-γ (Human IFN-γ ELISA Kit II, BD Biosciences; San Diego, CA, USA), and IL-4 (IL-4 ELISA kits, R&D Systems, Inc., Minneapolis, MN, USA) were used. Flow cytometry analysis was carried out by the above mentioned method.
4
Measuring CD4+ T Cell Activation
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Purified CD4+ T cells (5–8 × 105 cells/well in 96-well plates) from the PBMCs of healthy donors were co-cultured with RPE and LbL-RPE cells (5–8 × 103 cells/well: effector/target ratio = 500:1, and 100:1) for 48 h. T cell activation was evaluated by measuring IFN-γ production using ELISA (Human IFN-γ ELISA Kit II, BD, 550612).
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