Legendplex v8

Fluorochrome-conjugated anti-human monoclonal antibodies were used to identify human immune cells. Cells were incubated with appropriate dilutions of fluorescently labeled mouse anti-human monoclonal antibodies (Table S1) for 15 min at 4°C, washed with PBS containing 1% (w/v) bovine serum albumin (Sigma-Aldrich), and analyzed using FACS Fortessa or Verse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For each analysis, a live gate with white blood cells or lymphocytes was used based on human CD45 expression. The data were analyzed using FlowJo, PRID:SCR_008520 (BD Biosciences).
The culture supernatants were collected. Debris was removed by centrifugation (300 ×g 5 min at 4°C). Cytokine quantification was performed using LEGENDplex v8.0 (BioLegend, San Diego, CA, USA). Briefly, 25 µL of the supernatant was mixed with 25 µL of capture beads and incubated for 2 h at 25 °C. The beads were washed, mixed with detection antibodies, and incubated for 1 h at 25 °C. Streptavidin–phycoerythrin was added, and the mixture was incubated for 30 min at 25 °C. The beads were washed and analyzed using a FACSVerse flow cytometer (BD Biosciences). Data (pg/mL) were analyzed using LEGENDplex v8.0.

Fluorochrome-conjugated anti-human monoclonal antibodies (mAbs) were used to identify human immune cells. The cells were incubated with appropriate dilutions of fluorescently labeled mAbs for 15 min at 4°C and were then washed with PBS containing 1% (w/v) Bovine Serum Albumin (BSA) Sigma-Aldrich, St. Louis, USA). The cells were analyzed using FACS Fortessa, or Verse (BD Bioscience, Franklin Lakes, NJ, USA). For each analysis, the live gate with white blood cells or lymphocytes was further gated based on hCD45 expression. The data were analyzed using FlowJo™ v10 (BD Bioscience). The mouse anti-human mAbs used in this study are shown in Table S2.
Culture supernatants were collected, and the debris was removed by centrifugation at 300xg at 4°C. Cytokine quantitation was conducted using LEGENDplex (BioLegend) according to the manufacturer’s instructions. Briefly, 25 µL of the supernatant was mixed with 25 µL capture beads and incubated for 2 h at 25°C. The beads were washed, mixed with detection antibodies, and incubated for 1 h at RT. Subsequently, streptavidin-phycoerythrin was added, and the mixture was incubated for 30 min at 25°C. Finally, the beads were washed and analyzed using FCM. Analysis was performed using the BD FACSVerse™ Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed in pg/mL using LEGENDplex™ V8.0 (BioLegend, San Diego, USA).

Supernatants of the cultured cells were collected for cytokine quantitation using bead-based multiplex LEGENDplex (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, 25 µL of supernatant was mixed with 25 µL of capture beads and incubated for 2 h at 25 °C. The beads were then washed, mixed with detection antibodies, and incubated for 1 h at room temperature. Subsequently, streptavidin–phycoerythrin was added, and the mixture was incubated for 30 min at room temperature. Finally, the beads were washed and analyzed by FCM. The cytokines IL-1β, interferon (IFN)-α, IFN-γ, tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-18, IL-22, IL-23, and IL-33 were quantified. Analysis was performed using the BD FACSVerse^TM Flow Cytometer (BD Biosciences). The data were analyzed in picogram per milliliter using LEGENDplex™ V8.0 (BioLegend). IFN-γ was quantified using ELISA with serially diluted samples using OptEIA^TM Human IFN-γ ELISA Set (BD Biosciences) according to the manufacturer’s instructions.