Trustain fcx blocking solution

To confirm the identity of specific cell types expressing ambiguous markers at the RNA level, during the last experimental run (run 16), half the cells from each experimental condition were used to perform CITE-seq, according to the manufacturer’s instructions (10x Genomics). PBMCs were washed, resuspended in chilled 1% BSA in phosphate-buffered saline and incubated with human TruStain FcX blocking solution (BioLegend) for 10 min at 4 °C. Cells were then stained with a cocktail of TotalSeq-B antibodies (BioLegend) previously centrifuged at 14,000g for 10 min (Supplementary Table 3b). The cells were incubated for 30 min at 4 °C in the dark and were then washed three times. Cell density was then adjusted to 1,000 viable cells per µl in 1% BSA in phosphate-buffered saline. We generated scRNA-seq libraries and cell protein libraries (L131–L134) with the Chromium Single Cell 3′ Reagent Kit (v.3.1), using the Feature Barcoding technology for Cell Surface Proteins (10x Genomics).

Cells were washed and resuspended at a density of ≤10⁶ cells/25 µl flow buffer, and Fc receptors were blocked by adding 10 µg ml^–1 TruStain FcX™ blocking solution (BioLegend). Cells were then stained in a 50 µl final volume in 96-well round-bottom plates (Corning) covered for 30 min at 4°C and washed twice with flow buffer (1× PBS, 2% FBS, 1% sodium azide) before resuspension in 150 µl of BD™ Stabilizing Fixative (BD Biosciences) and transfer to polystyrene tubes (12×75 mm) (Becton Dickinson). A total of 0.5×10⁵ to 1×10⁵ events were acquired on a BD FACSCanto™ flow cytometer with FACSDiva™ software (BD Biosciences) and analyzed by FlowJo. Fluorophore-conjugated antibodies were APC-CD45 (BD Biosciences) and BV421-CD11c, BV421-CD8, FITC-Ly6C, PE-F4/80, PE-CD31, PerCP-CD3, PerCP-CD19, PE/Cy7-Ly6G, APC/Cy7-CD11b, and APC/Cy7-CD4 (BioLegend) (antibody details in Table S6).