Opteia mouse ifn γ elisaset

The contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum were detected using kits from Nanjing Jiancheng Bioengineering Institute. The serum interferon gamma (IFN-γ), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 12 p70 (IL-12 p70) and tumor necrosis factor alpha (TNF-α) were detected using the OptEIA Mouse IFN-γ ELISA Set, OptEIA Mouse IL-4 ELISA Set, OptEIA Mouse IL-10 ELISA Set, OptEIA Mouse IL-12 (p70) ELISA Set and OptEIA Mouse TNF-α ELISA Set (BD Biosciences, USA).

The supernatants obtained during the degranulation experiment performed with the in vitro-expanded murine NK cells were collected and the production of IFN-γ (produced by the NK cells) was analyzed by the commercial ELISA kit BD OptEIA Mouse IFN-γ ELISA Set following the manufacturer's instructions.

Cecum and spleen homogenates were centrifuged twice for 10 minutes at 13,000
g, and the supernatants were collected, diluted 1:2 in
DPBS, and stored at -20°C. Levels of interferon-γ (IFN-γ) were determined by
enzyme-linked immunosorbent assays (ELISAs) using BD OptEIA Mouse IFN-γ ELISA
set (BD Biosciences) according for the manufacturer’s instructions. IFN-γ levels
were normalized to the weight of the organs.

Three days poststimulation with Spike_RBD 15-mer peptides, cell supernatants were collected and used for ELISA to measure IFNγ, IL-6, IL-2, and TNFα production. IFNγ, IL-2, IL-6, and TNFα were measured using the BD OptEIA Mouse IFN-γ ELISA Set (BD Biosciences, 555138), BD OptEIA Mouse IL-6 ELISA Set (BD Biosciences, 555240), BD OptEIA Mouse IL-2 ELISA Set (BD Biosciences, 555148), and Mouse TNFα Uncoated ELISA kit (Invitrogen, 88-7324-22) per manufacturer's protocol.

Single cell suspensions of splenocytes were prepared from the excised spleens of mice vaccinated once with DCs pulsed with P9 or P17 (three mice per peptide for a total of six mice). Spleen cell preparations from each individual mouse were divided in half for either CD4^pos depletion or CD8^pos depletion. The MidiMACS system (MidiMACS separator, LS Columns, CD8 (TIL) Microbeads, and CD4 (L3T4) Microbeads) was used for depletion via manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Following separation, the preparations were analyzed via flow cytometry to ensure successful depletion and used in an in vitro recall assay for the vaccinating peptide (P9 or P17). Recall of antigen was assessed from 24 h stimulated culture supernatants using BD OptEIA mouse IFNγ ELISA set (BD Biosciences, San Diego, CA, USA) via manufacturer’s instruction. A stimulation index (IFN-γ output in stimulated divided by unstimulated cells) was determined for each individual mouse and then averaged.