Anti cd16 pe clone 3g8

Monocyte subpopulations were isolated from peripheral blood mononuclear cells (PBMC) obtained from SpA patients. PBMC were isolated from EDTA-treated whole peripheral blood by the standard Pancoll human (PAN-Biotech, Aidenbach, Germany) density gradient centrifugation. PBMC were washed in PBS (Sigma-Aldrich, Saint Louis, USA) and then monocyte subsets (classical—CD14⁺⁺CD16⁻, intermediate—CD14⁺⁺CD16⁺ and non-classical—CD14⁺CD16⁺⁺) were isolated using flow cytometry cell sorting. The following monoclonal antibodies (mAbs) were used to stain monocytes: anti-CD14-FITC (clone MφP9, BD Biosciences, San Jose, CA, USA), anti-CD16-PE (clone 3G8, BD Biosciences) and anti-HLA-DR-PerCP (clone L243, BD Biosciences), in 1:25 dilution v/v stained and gated as previously described by us and others^{47 (link),48 (link)}. The stained monocytes were then incubated for 30 min at 4 °C after which they were sorted using the FACSAria II cell sorter (BD Biosciences). Sorter was equipped with 488 nm laser for excitation of FITC, PE and PerCP. The following band-pass filters were used for the measurement of fluorescence: 530/30 for FITC, 582/42 for PE and 695/40 for PerCP. After isolation, the cells were washed in PBS, centrifuged for 10 min at 350×g and kept frozen at − 80 °C until RNA isolation.

Peripheral whole blood samples were collected in sterile BD Vacutainer^® EDTA (ethylenediamine-tetraacetic acid) blood collection tubes (BD Biosciences). The samples were incubated in BD Trucount™ tubes (BD Biosciences) with the following monoclonal antibodies to identify lymphocyte sub-populations (BD Biosciences): anti-CD45-PerCP (clone 2D1), anti-CD3-FITC (clone UCHT-1), anti-CD4-APC (clone RPA-T4), anti-CD8-PE (clone RPA-T8), anti-CD16-PE (clone 3G8), anti-CD19-APC (clone HIB19), anti-CD56-PE (clone NCAM16.2). The following antibodies were used for monocyte sub-populations (BD Biosciences): anti-CD45-APC (clone 2D1), anti-HLA-DR-PerCP (clone L243), anti-CD14-FITC (clone M5E2), and anti-CD16-PE (clone 3G8). The samples were incubated at 4 °C for 30 min and then were treated with FACS Lysing Solution (BD Biosciences) until the erythrocytes were lysed and the cells were immediately processed in the FACSCanto flow cytometer (Becton–Dickinson Immunocytometry Systems, Palo Alto, CA) along with 10,000 beads per tube. The results were analyzed with FACSDiva Software (BD Biosciences) and the absolute numbers of lymphocytes and monocytes subsets were calculated on the basis of bead counts. Monocyte populations were classified as classical (CD14++ CD16−), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++).12 (link)