Il 12 elisa max standard set

The IFN-γ-secreting splenocytes were measured at 7, 17, and 45 dpi using a mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Z_EDIII (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB). To measure secreted cytokines, splenocytes (5 × 10⁵/well) were stimulated with Z_EDIII (1 μg/ml) for 48 h and the supernatant of splenocytes was collected. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate. To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (clone 145-2C11, BD Biosciences), and anti-mouse CD4 (clone RM4-5, BD Biosciences) or anti-mouse CD8 (clone 53–6.7, BD Biosciences) as described previously [27 (link)]. Cell phenotype was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

The interferon (IFN)-γ secreting splenocytes were measured at 7, 17, and 45 days after the first immunization using mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Env_M (1 μg/ml) or Env_Z (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB).
For cytokine analysis, 5 × 10⁵ splenocytes/well were stimulated with Env_M (1 μg/ml) or Env_Z (1 μg/ml). The media from splenocyte cultures were collected after 48 h incubation. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate.
To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (fluorescein isothiocyanate, BD Biosciences), and anti-mouse CD4 (phycoerythrin [PE], BD Biosciences) or anti-mouse CD8 (PE, BD Biosciences). Cell phenotype was analyzed using a FACSCalibur flow cytometer (Becton Dickinson).