Cd16 fitc clone 3g8

A series of commercially available human monoclonal antibodies that cross-react with NHP mononuclear cells were used in flow cytometry analyses, as described previously [11 (link), 13 (link), 14 (link)]. Briefly, 100 μL of whole blood from each sample was added to each 12-mm × 75-mm polystyrene test tube (Falcon, Lincoln Park, NJ) containing panel of monoclonal antibodies CD3 Percp (clone SP-34), CD8 PE (clone SK1), CD16 FITC (clone 3G8) and CD20 APC (clone L27) (all from BD Biosciences, San Diego, CA) and incubated for 15 min at room temperature in the dark. Red blood cells were lysed with 1× FACS lysing solution (Becton Dickinson, San Diego, CA), diluted according to the manufacturer's instructions. The samples were washed thoroughly in 1× phosphate-buffered saline (PBS) by centrifugation; cell sediments were then suspended in 1% paraformaldehyde buffer (300 μL), and cells were acquired on a 4-color flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA). Lymphocytes that were gated on forward scatter versus side scatter dot plot were used to analyze CD3⁺, CD4⁺(CD3+CD8-),CD8⁺ (CD3+CD8+) T-CD16+ (NK cell), CD3+CD16+ (NKT) and CD20⁺ B-cell lymphocyte subsets with use of FlowJo software (Tree Star, Inc., Ashland, OR).

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.