Anti cd16 pe cy5

Peripheral blood from healthy donors was obtained from the Red Cross Blood Center. We followed the guidelines of the Red Cross, and all of the methods and protocols used in this study with human peripheral blood were approved by the Institutional Review Board of the Red Cross. Based on the Red Cross guidelines, informed consent for study participation was obtained and donor information could not be provided. For macrophage-differentiation and osteoclast-formation experiments, human monocytes were isolated from peripheral blood to achieve greater than 95% purity based on negative selection using the RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada). The purity of the isolated monocytes was analysed by flow cytometry using anti-CD14-APC (BioLegend, San Diego, CA, USA), anti-CD16-PE-Cy5 (BioLegend), and anti-CD3-APC-Cy7 (BioLegend) antibodies in phosphate-buffered saline plus 1% foetal bovine serum (FBS) on ice for 10 min. Cells were then washed and analysed. Staining data were collected using a FACSCanto II Cytometer (BD Biosciences). To set the gates for defining positive and negative cells in the multicolour staining, samples were stained with a mixture of all antibodies.

Immediately after collecting WBC, we rinsed the cell pellet with 300 μL PBS 1X (Sigma Aldrich, St. Louis, MO, USA) and centrifuged at 1800× g for 15 min at room temperature. After adding 5 mL ammonium-chloride-potassium (ACK) lysing buffer (Thermo Fisher Scientific, Vienna, Austria), we resuspended the cell pellet gently and incubated it for 5 min at room temperature. Then, we centrifuged at 1800× g for 10 min and discarded the supernatant, rinsing and resuspending the cell pellet twice with 300 μL PBS 1X. Next, we resuspended 4 × 10⁶ WBC in 50 μL cell staining buffer (BioLegend, Inc., San Diego, CA, USA), adding 5 μL True-Stain Monocyte Blocker^TM (BioLegend, Inc., San Diego, CA, USA) for 10 min on ice. Immediately after, we added anti-CD14 PE/Cy7, anti-CD16 PE/Cy5, Zombie UVTM dye (BioLegend, Inc., San Diego, CA, USA), and anti-HLA-DR BUV661 (BD Biosciences, San Jose, CA, USA) for 20 min in darkness at 4 °C. After centrifuging and rinsing with cell-staining buffer, we added 150 μL PBS 1X, acquiring 10,000 cell events per test in triplicate corresponding to the HLA-DR+ cell population on a BD Influx flow cytometer (BD Biosciences, San Jose, CA, USA), using the BD software^TM version 1.2. (BD Biosciences, San Jose, CA, USA).