Anti cd73 apc

Fluorescence-activated cell sorting analysis was performed using a BD LSRFortessa™ (BD Biosciences, San Jose, CA). The following antibodies were used to classify cells: anti-CD19-fluorescein isothiocyanate (FITC) (Miltenyi Biotec, Friedrich Gladbach, Germany), anti-CD3-allophycocyanin (APC), anti-CD4-APC, anti-CD4-phycoerythrin (PE), anti-CD24-peridinin chlorophyll protein complex-Cy 5.5 (PerCP Cy5.5), anti-CD25-APC, anti-CD27-PE, anti-CD39- brilliant violet 421 (BV421), anti-CD45-V500, anti-CD73-APC, anti-IFN-γ-BV421, and anti-IL-10-BV421 (all from BD PharMingen, Franklin Lakes, NJ), anti-CD80-APC, anti-CD86-APC monoclonal antibody (mAb) (Biolegend, San Diego, CA), Annexin V- FITC (BD PharMingen), and DAPI (Cell Biolabs, San Diego, CA). The CD19⁺CD24^hiCD27⁺ B cell subset was sorted using a Moflo XDP cell sorter (Beckman Coulter, Brea, CA) with 90% to 95% purities.

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.

Four week-old SD rats were sacrificed and the primary BMSCs were isolated from the femurs and tibias under a sterile condition. Following flushing the bone marrow cavity with DMEM-LG medium (Invitrogen Life Technologies, Grand Island, NY, USA) and removing large tissues with a 200-mesh nylon filter, the isolated bone marrow cells were cultured in DMEM-LG medium supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies), 1% penicillin and streptomycin (Invitrogen Life Technologies) at 37°C in an environment with 5% CO₂. The medium was refreshed every 3 days and cells were harvested by multiple digestions and passaged when cell confluence reached >80%. Cells of the fifth passage were collected and subjected to flow cytometry analysis. These cells were maintained in adipogenic, osteogenic and chondrogenic differentiation medium (Cyagen Biosciences, Sunnyvale, CA, USA) followed by Oil Red O staining, Alizarin Red S staining and Toluidine Blue staining, respectively. The surface markers were detected using a BD FACSC™ II flow cytometry system (BD Biosciences, San Jose, CA, USA). The antibodies utilized in this analysis included anti-CD44-PE, anti-CD73-APC, anti-CD90-FITC, anti-CD105-PerCP-Cy5.5, anti-CD11b-PE, anti-CD19-PE, anti-CD34-PE and anti-CD45-PE (BD Biosciences). Isotype-matched antibodies were used as controls. Cells were harvested between 3–5 passages.