Ecd anti human cd14

Whole peripheral blood from RA patients and HDs (100 μl) was incubated with ECD anti-human CD14 (Beckman Coulter, Indianapolis, IN, USA) and APC/Cy7 anti-human CD16 (BioLegend, San Diego, CA, USA). After lysis and Fixation/permeabilization (BD Cytofix/Cytoperm™ Fixation/Permeabilization solution Kit with BD GolgiPlug™; BD Biosciences, San Jose, CA, USA), cells were incubated either with PE anti-human TNF-α (Immunostep, Salamanca, Spain) or PE anti-human IL-6 (Immunostep) or primary antibody anti-human IKK (Abcam, Cambridge, UK) for 30 min at 4°C in the dark. Then, for IKK analysis, PE conjugated secondary antibody (Abcam, Cambridge, UK) was added for 30 min at 4°C. IgG isotypes were used as negative controls. Cells were washed and acquired on the flow cytometer FC 500 (Beckman Coulter).

Peripheral blood mononuclear cells from RA patients belonging to the second cohort were isolated through ficoll density gradient. Briefly, granulocytes and natural killer cells were removed using immunomagnetic labeling. Firstly, CD16⁺ monocytes were isolated through positive selection (using human anti-CD16, CD16⁺ monocyte isolation kit, MiltenyiBiotec, Bergisch Gladbach, Germany). Thereafter, the CD14⁺ monocytes were isolated from the eluted fluid using positive selection (human anti-CD14, CD14 microbeads, MiltenyiBiotec). Purity of each cell fraction isolated was tested by flow cytometry (FC500 Beckman Coulter), using ECD anti-human CD14 (Beckman Coulter), APC/Cy7 anti-human CD16 (BioLegend). Non-specific antibodies conjugated to ECD and APC/Cy7, respectively, were used as negative controls. Purity of isolated cells was always >90%. The percentage of CD14⁺CD16⁺ (intermediate) and CD14-CD16⁺ (non-classical) cells in CD16⁺ isolated fraction was 55.5 ± 6.2% and 40.3 ± 4.7%, respectively Datasheet 1.

Whole peripheral blood from RA patients and healthy donors (100 μl) was lysated with 2 ml of lysis buffer (VersaLyse; Beckman Coulter) for 10 min at room temperature in the dark. Cells were incubated with ECD anti-human CD14 (Beckman Coulter), APC/Cy7 anti-human CD16 (BioLegend) and FITC-Conjugated monoclonal antibody against human tissue factor (Sekisui Diagnostics, LLC Stamford, CT, USA) for 20 min at 4°C in the dark. IgG isotypes were used as negative controls. Cells were washed and acquired on the flow cytometer FC 500 (Beckman Coulter).