One day before the experiment, 2.0 × 105 HaCaT cells were cultured in 48-well plates. Cells were stimulated with 10 μg/mL LPS or 10 μg/mL Df for 24 h in 100 µL DMEM containing 0.5% FBS for 24 h. In 96-well plates, 2.0 × 104 monocyte-derived LCs were cultured and stimulated with 10 μg/mL LPS or 10 μg/mL Df for 24 h in 100 μL of RPMI 1640 containing 0.5% FBS for 24 h. After the 24-h stimulation, 0.5% Triton X-100 was added directly into the cell cultures and mixtures of cell suspension and lysate were collected. The mixtures were centrifuged at 14,000 rpm at 4°C for 5 min to remove cell debris. The mixtures of cell lysate and supernatant were used for the ELISA assay following the kit protocol.
Cytokine/chemokine multiplex array was performed using the Inflammation 20-Plex Human ProcartaPlex Panel (EPX200-12185-901, Invitrogen) and analyzed with a Bio-Plex 200 (BioRad) following manufacturer protocols. ELISA kits for CCL17 (DY364), MCP1 (DCP00), MDC (DMD00), IL-4 (D4050), and GM-CSF (DY215) were purchased from R&D Systems. PDGF-A ELISA kits (EHPDGF) were purchased from Thermo Fisher Scientific. Absorbance (excitation, 450 nm) was measured with a microplate reader (Infinite F200 Pro, Tecan). For reference, background absorbance was measured at 560 nm.
Cytokine/chemokine multiplex array was performed using the Inflammation 20-Plex Human ProcartaPlex Panel (EPX200-12185-901, Invitrogen) and analyzed with a Bio-Plex 200 (BioRad) following manufacturer protocols. ELISA kits for CCL17 (DY364), MCP1 (DCP00), MDC (DMD00), IL-4 (D4050), and GM-CSF (DY215) were purchased from R&D Systems. PDGF-A ELISA kits (EHPDGF) were purchased from Thermo Fisher Scientific. Absorbance (excitation, 450 nm) was measured with a microplate reader (Infinite F200 Pro, Tecan). For reference, background absorbance was measured at 560 nm.