Cd14 rd1 clone 322a 1

MDM from HD and patients with RA were obtained as explained before. Monocytes were isolated from 50 mL of venous blood from HD using Monocyte Enrichment Cocktail (Stem Cell Technologies) following the manufacturer’s instructions. Total monocytes (purity > 90%) were stained with CD14-RD1 (clone 322A-1; Beckman Coulter, CA) and CD16-FITC (clone 3G8; BD) antibodies for 15 min at 4 °C. CD16 − (purity > 95%) and CD16 + (purity > 90%) monocytes were sorted by using MoFlo XDP (Beckman Coulter, CA).
MDM were cultured for 6 h in the absence or presence of PRA-m/lEVs or PRA-m/lEV-ICs at a ratio monocyte:m/lEVs of 1:3. Enriched monocytes were cultured for 24 h as previously described [34 (link)], in the absence or presence of PRA-m/lEVs or PRA-m/lEV-ICs at ratio monocyte:m/lEVs of 1:3. Supernatants were stored at − 20 °C.

HUVEC and HMVEC-L were cultured with MPs and MPs-ICs from patients with SLE and RA for 24 h as previously stated. Next, the cells were washed with EBM-2 to remove excess vesicles. Monocytes were isolated from 50 mL of venous blood from healthy individuals using RosetteSep™ Human Monocyte Enrichment Cocktail (STEMCELL Technologies) according to the manufacturer’s instructions. Total monocytes (purity > 90%) were stained with 0.2 μM CFSE (Thermo Scientific) for 45 min at 37 °C, washed twice with PBS, and stained with CD14-RD1 (clone 322A-1; Beckman Coulter, Brea, CA, USA) and CD16-FITC (clone 3G8; BD Pharmigen™) antibodies for 15 min at 4 °C. Classical (CD16-, purity > 95%) and non-classical (CD16+, purity > 90%) monocytes were sorted by using MoFlo™ XDP (Beckman Coulter). CD16- and CD16+ monocytes were added to endothelial cells at a proportion 1:1, centrifuged at 200 g for 1 min, and cultured for 30 min at 37 °C. Non-adherent monocytes were removed by multiple washes with EBM-2. Adherent monocytes were visualized and counted in an inverted epifluorescence microscope (Nikon ECLIPSE TS 100; Nikon, Tokyo, Japan) using a ×40 objective in at least five different fields per experimental condition.