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Apc conjugated anti ifn γ antibody

Manufactured by BD

The APC-conjugated anti-IFN-γ antibody is a laboratory reagent used to detect and quantify the presence of interferon-gamma (IFN-γ) in biological samples. The antibody is conjugated with Allophycocyanin (APC), a fluorescent dye, allowing for the identification and analysis of IFN-γ-producing cells through flow cytometry or other immunoassay techniques.

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3 protocols using apc conjugated anti ifn γ antibody

1

Evaluating NK Cell Cytotoxicity Against Breast Cancer

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Purified NK cells (2.5×105) were cultured alone or with breast cancer cell lines in 96-well plates in a total volume of 200 μl. Monensin (1μM, eBioScience) was added to cultures. Cells were stimulated with tumor and Trastuzumab with the presence of different mAbs for 16 h at 37 °C with 5% CO2. Cells stimulated with phorbol-12-myristate-13-acetate (PMA) (100 ng/ml; Sigma) and ionomycin (1μM, Sigma) were used as positive controls. After incubation, cells were collected and blocked with human serum 100μl for 15 min at 4 °C. Then cells were labeled with PE-conjugated anti-CD56 (BD Pharmingen) antibodies for 30 min at 4 °C. After cell surface staining, cells were fixed and permeabilized with Fixation and Permeabilization Buffer (BD Pharmingen) and stained with APC-conjugated anti-IFN-γ antibody (BD Pharmingen). After washing with the staining buffer, the cells were resuspended in 300 μl cold staining buffer, followed by analysis with flow cytometry.
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2

Analyzing Antigen-Specific T-cell Responses

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Lymphocytes collected from the spleens of immunized mice were cultured for 18–20 h in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 50 nM β-mercaptoenthanol, 100 units/ml penicillin/streptomycin, 2 mM l-glutamine (Welgene), and 10 μg/ml of purified ScaA in 24-well, flat-bottomed culture plates (5 × 106 cells/well). After incubation, GolgiPlug (BD Biosciences) was added for 6 h. Lymphocytes were washed three times with ice-cold FACS buffer (PBS containing 1% bovine serum albumin and 1 mM EDTA). Cells were blocked on ice for 30 min with ultra-block solution containing 10% rat sera, 10% hamster sera, 10% mouse sera (Sigma) and 10 μg/ml of 2.4G2 monoclonal antibody (BD Pharmingen). Cells then stained with FITC-conjugated CD4 or PE.cy7-conjugated CD8 antibodies (BD Pharmingen) for 30 min at 4 °C. After surface CD4 or CD8 staining, cells were washed three times with ice-cold FACS buffer and subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Biosciences). Intracellular interferon-γ (IFN-γ) was stained using an APC-conjugated anti-IFN-γ antibody (BD Pharmingen) for 30 min at 4 °C. The stained cells were analyzed with a FACS LSRII flow cytometer (BD Biosciences). Data were analyzed by FlowJo software version 8.8.6 (FlowJo).
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3

Analyzing Cytokine Profiles in Colitis

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To determine the concentration of the cytokines including IL-2, IFN-γ, IL-4, IL-6, TNF, IL-17A and IL-10 in the colons and serum of the animals, the BD™ Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine Kit was used according to the manufacturer's instruction. Data were analyzed with FCAP Array software.
For flow cytometry assay, mesenteric lymph nodes (MLNs) and spleen single-cell suspensions were harvested by gently pushing cells through a 70-μm cell strainer with a syringe piston as previously described [34 (link)]. For Treg analysis, cells were stained with FITC conjugated anti-CD4 antibody at 4 ​°C in the dark for 20 ​min. They were fixed and permeabilized for 40 ​min in the dark, and finally stained with PE conjugated anti-FOXP3 antibody. For Th1/Th2/Th17 analysis, cells were first stimulated with a leukocyte activation cocktail in 5% CO2 at 37 ​°C for 6 ​h. Subsequently, they were stained with FITC conjugated anti-CD4 antibody (BD Biosciences) for 30 ​min in the dark, and then fixed and permeabilized for 40 ​min. Finally, they were stained with APC conjugated anti–IFN–γ antibody (BD Biosciences), BV605 conjugated anti-IL-17A antibody (BD Biosciences), and PE conjugated anti-IL-4 antibody (BD Biosciences). After washing with permeabilization buffer (BD Biosciences), the stained cells were analyzed by flow cytometer.
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