Cd163 bv421

For phenotypic analysis of myeloid cells in 7 SCC and 3 AC tumors, multi-color flow cytometry was carried out using the LSR Fortessa X-20 (BD). Samples were freshly collected as described previously (33 (link), 34 (link)). A total of 200,000 cells per tumor sample were stained using the following directly labeled surface antibodies: CD1a-PE (1:50, BD), CD14-PerCP-Cy5.5 (1:20, BD), CD11c-APC (1:100, BD), CD1c-PE-Cy7 (1:100, Biolegend), CD45-AF700 (1:200, Biolegend), PD-L2-BV711 (1:25, BD), PD-L1-BV786 (1:25, BD), CD80-FITC (1:50, BD), and CD163-BV421 (1:70, BD). Data were visualized in t-Distributed Stochastic Neighbor Embedding (t-SNE) density plots generated in FCS express 6 (De Novo software).

To induce differentiation to macrophages, THP-1 cells were pre-treated for 24 h with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and further incubated in fresh RPMI 1640 medium (Gibco) for 24 hours. On day 2, differentiated macrophage cells were stimulated with M1 cytokines (20 ng/mL IFN-γ, 1 μg/mL LPS; Peprotech) or M2 cytokines (20 ng/mL IL-4, 20 ng/mL IL-13; Peprotech), with or without WJ-MSCs, in a 12-well transwell plate (0.4 μM pore size, Corning, Lowell, MA, USA). WJ-MSCs were cultured at a density of 1X10⁵ in the upper layer, while THP-1 cells were placed at a density of 5 × 10⁵ in the lower layer in RPMI 1640 medium supplemented with 10% FBS. After co-culture for 48 h, the cells were stained with fluorescence-labeled human monoclonal antibodies against CD14-APC-H7, CD80-PE-Cy7, and CD163-BV421 (BD Biosciences) and analyzed by flow cytometry.