Cd64 phycoerythrin pe

The following antibodies were used to label the CD markers: CD16-Alexa Fluor 700 (BioLegend, San Diego, CA, USA), CD63-peridinin chlorophyll protein (PerCP)-Cy5.5 (BioLegend), CD66-allophycocyanin (APC) (eBioscience, Santa Clara, CA, USA), CD14 PE-Cy7 (BioLegend), CD18-brilliant violet 421 (BV421) (BD), CD11b-APC-Cy7 (BioLegend) and CD64-phycoerythrin (PE) (BD). SONY SA3800 flow cytometer was used to acquire the data, and the channel voltages were calibrated manually with rainbow beads to normalize sample acquisition on different days. Compensation was performed with single-stained OneComp eBeads (eBioscience). Data were analysed by FlowJo software (v10; Tree Star), and the geometric mean fluorescence intensities (MFIs) were determined for each CD marker and H3Cit, as mentioned previously by Fine et al. [13 (link)]. Gating was then performed, as described by Fine et al. [13 (link)] (Figure A1), and the geometric mean fluorescence intensities were used for data analysis.

BM-MSCs (from 8 donors between P3 and P7) were harvested, washed, and resuspended in PBS-sodium azide buffer (0.1%) at a density of 10 × 10⁶ cells/ml. Cell suspensions (0.5 × 10⁶ cells/50 µl) were incubated with 3 to 5 µl of monoclonal antibodies for 25 min at 4 °C in the dark. The following anti-human antibodies were used: CD14-fluorescein isothiocyanate (FITC; BD Biosciences), CD34-allophycocyanin (APC; BD Biosciences), CD45-peridinin chlorophyll protein complex (PerCP; BD Biosciences), CD64-phycoerythrin (PE; BD Biosciences), CD73-PE (BD Pharmingen), CD146-PE (BD Pharmingen), HLA-DR-FITC (BD Biosciences), CD29-FITC (Beckman Coulter), CD44-PE (Beckman Coulter), CD90-PE (Beckman Coulter), and CD105-FITC (ABd Serotec). The respective mouse isotype antibodies served as controls. The cells were then washed and resuspended in 300 μL PBS-azide. Acquisitions were performed in a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed using FlowJo Software (TreeStar).