Antigen-specific spleen cell IFN-γ secretion was also assayed by ELISPOT as described earlier. Briefly, 96-well flat-bottomed nitrocellulose plates (MAHA S4510, Millipore, Billerica, MA) were incubated overnight at 4°C with 50 µl of capture purified anti-mouse IFN-γ (15 µg/ml; BD Pharmingen, Erembodegem, Belgium) in phosphate-buffered saline (PBS) and then saturated with 200 µl/well of RPMI-complete medium 2 h at 37°C. 180 µl of spleen lymphocytes (pool of four mice per group) were added at a cell concentration of 4.106 cells/ml in the presence or absence of 20 µl proteins (5 µg/ml) and plates were incubated for 48 h at 37°C, 5% CO2. After extensive washing, plates were incubated 2 h at 37°C, 5% CO2 with 50 µl of biotinylated rat anti-mouse IFN-γ (2 µg/ml) (BD Pharmingen), washed and incubated for 45 min at 37°C, 5% CO2 with alkaline phosphatase labelled ExtrAvidine (Sigma-Aldrich, Bornem, Belgium). After washing, spots were revealed with Bio-Rad (Hercules, CA) alkaline phosphatase conjugate substrate kit, following the manufacturer's instructions and plates were analysed on a Bioreader-3000 LC (BioSys, Germany). Results are shown as mean spot-forming cells (SFC) per million lymphocytes.
Biotinylated rat anti mouse ifn γ
Manufactured by BD
Sourced in Netherlands
Biotinylated rat anti-mouse IFN-γ is a laboratory reagent used for the detection and quantification of mouse interferon-gamma (IFN-γ) in various biological samples. It is a specific antibody that has been labeled with biotin, a small molecule that can be used to enhance signal detection.
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Lab products found in correlation
3 protocols using biotinylated rat anti mouse ifn γ
1
IFN-gamma ELISPOT Assay for Antigen-Specific T-cell Response
2
Quantifying IFN-γ in Reo-infected mouse cells
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Sorted Reo μ1133–140 Tm+ or Reo μ1422–430 Tm+ cells (2,000 cells/well of a round-bottom 96-wells plate) were cocultured with PMA (20 ng/mL) and ionomycin (1 μg/mL) or Reo-infected TC1 cells (20,000 cells/well). In some wells, NAb-containing plasma from Reo-preexposed mice (1:1,000 dilution) was added. After 48 hours of incubation, supernatants were harvested. For ELISA, Nunc MaxiSorp plates (Corning) were coated with purified rat anti-mouse IFNγ (BD Pharmingen, #551309) in sodium carbonate/sodium bicarbonate coating buffer (pH 9.6) overnight at 4°C and then blocked with PBS/1% BSA/0.05% Tween-20 (Merck) for 1 hour at 37°C. After washing with wash buffer (PBS/0.05% Tween-20), 100 μL of supernatant was added and incubated for 2 hours at RT. The standard curve was prepared using recombinant mouse IFNγ (BioLegend, #575302). After washing, biotinylated rat anti-mouse IFNγ (BD Pharmingen, #551506) was applied for 1 hour at RT, followed by poly-Streptavidin–HRP conjugate (Sanquin, the Netherlands, #M2051) for 1 hour at RT. After washing, 50 μL of TMB (3,3′,5,5′-Tetramethylbenzidine; Sigma–Aldrich, #T0440) was added and the reaction was quenched by the addition of 50 μL 2 mol/L H2SO4 (Merck). Absorbance was measured at 450 nm using a SpectraMax iD3 multi-mode plate reader (Molecular Devices).
3
CD8 T Cell IFN-γ ELISPOT Assay
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CD8 T cell enzyme-linked immunospot was performed as described (Sivaganesh et al., 2013 (link)). Briefly, purified CD8 T cells were mixed with irradiated BALB/c stimulator splenocytes and added to Multiscreen HTS filtration system plates (Millipore) that had been coated with anti-mouse interferon-γ (IFN-γ; BD Pharmingen) in 0.1 M bicarbonate buffer (pH 9.6). Plates were incubated at 37°C and 5% CO2 for 20 hr, and after washing, spots were developed with biotinylated rat anti-mouse IFN-γ (BD Pharmingen), followed by streptavidin-horseradish peroxidase and the substrate, H2O2, together with the 3-amino-9-ethylcarbazole color indicator. Plates were read (Autoimmun Diagnostika), and data were expressed as spot counts per 106 responder CD8 T cells for each well.
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