Cd14 af488

Phospho-STAT1 staining: Whole blood from individuals with DS and healthy controls were subject to Ficoll gradient to collect the mononuclear cell layer (PBMC) and frozen. For direct stimulation, thawed PBMCs were incubated in complete RPMI overnight, washed, stained with live-dead in PBS for 10 min at 37°C, and finally were stimulated for 15 min with indicated amounts of IFNα2b. For staining, cells were washed 2 times with 0.5% BSA and surface-stained on ice for 30 minutes (CD16-AF647, CD14-AF488, CD3-BV510, CD19-AF700, CD56-BV711, all from Biolegend) followed by fixation/permeabilization in 90% ice-cold methanol and staining with PE anti-phospho-STAT1 Y701 (1:25, BD).
IFNAR1 and IFNAR2 staining: hTERT-immortalized fibroblasts were scraped off in 5mM EDTA, washed, and stained with live-dead in PBS for 30 min on ice. They were then washed and stained with anti-IFNAR1 (clone AA3, courtesy of Sandra Pellegrini in Supplemental Fig 1A and clone MARI-5A3 from Millipore Sigma for Supplemental Fig 3A) or anti-IFNAR2 (PBL) for 2 hours on ice. The cells were washed and stained with biotin-conjugated rat anti-mouse IgG (H+L) (Thermo Fisher) for 40 min on ice, followed by PE-conjugated Streptavidin for 10 min on ice.
Flow cytometry was acquired on a Cytek Aurora or BD LSR Fortessa, and data were analyzed with Cytobank (https://www.cytobank.org/).