Legendplex mouse th cytokine panel 13 plex

The murine macrophage cell line RAW264.7 was purchased from ATCC. RAW264.7 cells were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C under 5% CO₂. The cell density was adjusted to 2 × 10⁵ cells/ml in 12-well plates. When the cell confluence reached about 50%, the culture medium was replaced with fresh RPMI-1640 medium containing different concentrations of CARDS toxin (0, 5, and 50 μg/mL). After 48 h of incubation, the cells were harvested and the levels of 13 cytokines including TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, and IL-22 in the supernatant were measured using LEGENDplex™ Mouse Th Cytokine Panel (13-plex) (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions.

Cytokine production in undiluted mouse nasal tissue homogenates (n = 8/group) or supernatants of stimulated splenocytes (n = 4/group) were determined with LEGENDplex™ Mouse Th Cytokine Panel (13-plex, BioLegend, London, UK). Except for reducing the used volumes described in the manual by half, the assay was performed according to manufacturer’s instructions. Flow cytometry was conducted with a BD LSR II (BD Biosciences, Heidelberg, Germany). The data were acquired by BD FACSDiva™ (BD Biosciences) and analyzed with LEGENDplex™ Data Analysis Software (BioLegend, London, UK).