WT and apoE-/- mice or WT and lrp8-/- mice, were inoculated with either WT B16 or apoE-/- B16 cells, with anti-CTLA4 antibody on day 0. These vaccinated splenocytes (VS) were harvested on day 7 and co-cultured with either WT B16 or apoE-/- B16 cells for 48hr, following which IFNγ levels in media were compared with ELISA assay. To set up co-culture, a total of 5 × 105 freshly isolated mouse T cells or splenocytes were plated in a volume of 600 μl per well of 24-well plates, then they were co-cultured with 5 × 104 B16 cells and stimulated with or without CD3/CD28 Dynabeads. Cells were exposed to apoE agonist COG133 at 0, 0.3, 3, 9, 15, 30 µM or human anti-APOE antibody at 1 µg/ml, 10 µg/ml and 20 µg/ml at 37°C under 5% CO2 for 24 hr or 48 hr. Supernatants were collected from triplicate wells, and IFNγ was assayed using the mouse uncoated IFNγ ELISA kit from Invitrogen (Carlsbad, CA). Readings were measured at 450 nm using the EnSpire 2300 Multilabel plate reader (Perkin Elmer, Waltham, Massachusetts, US).
Ifnγ mouse uncoated elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IFNγ Mouse Uncoated ELISA Kit is a laboratory tool used to measure the concentration of interferon gamma (IFNγ) in mouse biological samples. It is a quantitative enzyme-linked immunosorbent assay (ELISA) that employs the sandwich ELISA technique.
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Lab products found in correlation
Enspire 2300 multilabel plate reader, by PerkinElmer (1 mentions)
Enspire 2300 multilabel, by PerkinElmer (1 mentions)
Urea, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using ifnγ mouse uncoated elisa kit
1
Tumor Immunogenicity and T Cell Responses
2
Splenocyte-B16 Coculture IFNγ Assay
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A total of 5×105 splenocytes were cocultured with 5×104 B16 cells for 24 hours or 48 hours. Supernatants were collected from triplicate wells, and IFNγ was assayed using the mouse uncoated IFNγ ELISA kit from Invitrogen (Carlsbad, California, USA). Readings were measured at 450 nm using the EnSpire 2300 Multilabel plate reader (Perkin Elmer, Waltham, Massachusetts, US).
3
Immune Response Modulation Protocol
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CHP was purchased from NOF Corporation (Tokyo, Japan). Phosphate-buffered saline (PBS), ATCC-modified RPMI 1640 medium, fetal bovine serum (FBS), 2-mercaptoethanol, and antibiotic-antimycotic were purchased from Gibco (Carlsbad, CA, USA). G418 was purchased from Nacalai Tesque (Kyoto, Japan). EndoGrade OVA was purchased from Hyglos GmbH (Bernried, Germany). InVivoMAb anti-mouse PD-1 antibody (clone J43) was purchased from BioXCell (West Lebanon, NH, USA). Urea was purchased from Wako (Osaka, Japan). Slide-A-Lyzer™ Dialysis Cassettes (10 K MWCO, 3 mL) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IFN-γ Mouse Uncoated ELISA Kit was purchased from Invitrogen (Carlsbad, CA, USA). CpG with phosphorothioate modification was purchased from FASMAC (Kanagawa, Japan). PE-conjugated anti-mouse CD279 (PD-1) antibody (RMP1-30) and APC-conjugated anti-mouse CD8 antibody (53-6.7) were purchased from BioLegend (San Diego, CA, USA).
4
IFN-gamma ELISA Quantification Protocol
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The assay was performed with IFNγ Mouse Uncoated ELISA Kit (Invitrogen) as per the manufacturer's protocol. Briefly, the ELISA plates were coated overnight with capturing antibody. After blocking the plates, samples and standards were added and incubated for 2 hours at room temperature. Cytokine amounts were detected by anti-IFNγ detection antibody followed by the addition of HRP-conjugated streptavidin and 1x Tetramethylbenzidine substrate solution. The quantification was done as per the protocol using the standard curve.
5
IFNγ ELISA Quantification Protocol
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