THP-1 Macrophage Co-Culture Protocol

THP-1 monocyte cells (tib-202tm; ATCC) were cultured in RPMI-1640 medium (12633012; Thermo Fischer) with 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum (FBS). THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce macrophage phenotype as assessed by adherence to tissue culture flask and expression of CD11b integrin (Figure S2). For co-culture, we followed an established protocol^{16 (link),22 (link)}. Briefly, ABCB5+ DSCs or donor-matched ABCB5− fractions were plated at 2x10⁴ cells per well in 24-well plates in 0.5 ml Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM L-glutamine. After 24 hours, THP-1 derived macrophages were seeded on top or in transwell inserts (CLS3464; Corning) at 1×10⁵ cells per well. Co-cultures were incubated with 50 U/ml recombinant human interferon-gamma (IFN-γ) (285-IF-100; R&D Systems) for 24 hours and then stimulated with 20 ng/ml LPS (L3755; Sigma-Aldrich) and 50 U/ml IFN-γ for another 24-hour period before supernatants were harvested and analyzed by enzyme linked immunosorbent assay (ELISA) for IL-1RA (DRA00B; R&D Systems). IL-1RA levels were analyzed between conditions with unpaired t-tests.