Anti cd14 fitc clone mφp9

In 30 CLL cases, CD14+ cells were selected into two populations due to the Tie2 expression. Tie2-positive and Tie2-negative monocytes were sorted using BD FACSAria II flow cytometer (BD Biosciences; Franklin Lakes, NJ, USA). PBMCs were labeled with anti-CD14 FITC (Clone MφP9, Cat No.: 347493; BD Biosciences, Franklin Lakes, NJ, USA) and anti-CD202b (Tie2/Tek) PE (Clone 33.1, Cat No.: 334206; BioLegend, San Diego, CA, USA). The purity of the sorted populations was confirmed by flow cytometry and reached more than 97%. Purified fractions were used for RNA isolation.

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.