Legendplex human th cytokine panel

γδ T cells (5 × 10⁴/well) freshly isolated from PBMCs by magnetic sorting were stimulated with BrHPP in IL-2-containing medium in the presence or absence of pVC. On day 8, the supernatants were collected and assessed for cytokine content with a LEGENDplex^TM human Th cytokine panel (BioLegend). Based on these results, levels of IFN-γ and IL-13 were additionally quantified by ELISA (Duoset; R&D Systems, Wiesbaden, Germany).

Plasma samples were evaluated for the abundance of interleukin 2 (IL2) and interferon gamma (IFNG, best known as IFNγ) with the LEGENDplex™ Human Th Cytokine Panel (BioLegend, #740001), as previously described [15 (link)]. Data were analyzed with the LEGENDplex™ Data Analysis Software (BioLegend).

Levels of IFN-γ, TNF-α, IL-2, IL-6, and IL-10, in human T-cell assay supernatants were measured using LEGENDplex human Th Cytokine Panel (Biolegend) according to the manufacturer’s instructions. Analyses were performed using FACSCanto (Becton Dickinson) and analyzed with LEGENDplex Data Analysis Software Suite (Qognit).

Ad-MSCs were seeded in 6-well plates at a concentration of 1 × 10⁵ cells/well. At 4 h post-transfection, supernatants were collected, and secreted PGE₂, TGFβ1, IL8, and IFNα were quantified by ELISA (R&D System, Minneapolis, MN, USA). Secreted IL6, IFNγ, and TNFα were quantified by flow cytometry using the LEGENDplex™ Human Th Cytokine Panel (Biolegend, CA, USA) following the manufacturer’s protocol.

The concentration of 12 cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17 A, IL-17 F, and IL-22) in the culture supernatants of PBMCs and mouse serum was quantified by LEGENDplex Human Th cytokine panel (BioLegend, San Diego, CA, USA) on the flow cytometer (BD LSRFortessa, Franklin Lakes, USA) under close compliance with the manufacturer’s guidelines. The experiments were repeated in triplicate with three replicates each.

T-cell lines, at 0.5 × 10⁵ cells/well, were incubated for 24 h with the individual peptides (1 μM) in round-bottom 96-well plates. Cell-free culture supernatants were harvested from the stimulated T-cell lines and analyzed using a bead-based multiplex immunoassay (MIA), quantitating levels of IFN-ɣ, TNF-α, IL-2, IL-4, IL-5, IL-13, IL-10, IL-22, IL-6, IL-9, IL-17A, and IL-17F (LEGENDplex human Th cytokine panel, 741028; BioLegend) according to the manufacturer’s instructions and using FACSCanto II (BD). DMSO (an equimolar amount of DMSO as used for peptide stimulations) and PHA (Sigma, 1 µg/mL) were used as negative and positive controls, respectively. For analysis, the online cloud-based program, the LEGENDplex™ Data Analysis Software Suite, was used. Background signal from negative DMSO controls was subtracted from total concentration (pg/mL) per cytokine induced by antigen-specific stimulation. If the background signal was below the threshold for detection, the detection threshold concentration was subtracted from the measured cytokine concentration induced by specific stimulation.

The levels of INF-γ and other cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, and TNF-α) were evaluated in supernatants obtained from 1:1 (E:T) ratio co-cultures after 24 h using the LEGENDplex™ Human Th Cytokine Panel (BioLegend).