Cd14 apc cy7

Human monocytes were cultured alone or with mf or three different CMFDA-labeled cancer cell lines (green; FITC channel) as mentioned above. After 48 hrs, cells were harvested, washed with PBS and incubated with 10 μl human IgG (10 mg/ml; Sigma-Aldrich, St. Louis, MO) for 10 min at 4°C to inhibit nonspecific binding through FcγRs and then incubated with marker-specific mAb conjugated with PDL2- APC (eBioscience, Cat No. 17-5888-42), VCAM1(Vascular cell adhesion molecule-1)-APC (Biolegend, Cat No. 305810), CD14-APC Cy7 (eBioscience, Cat No. 47-0149-42), CD45-Pacific blue (Biolegend, Cat No. 304022), CD206 (Mannose receptor)-Percp efluor 710 (eBioscience, Cat No. 46-2069-42), PDL1-PE-Cy7 or PDL1-APC (eBioscience, Cat No. 25-5983-42 and Cat No. 17-5983-41 respectively), or CD163-PE (eBioscience, Cat No. 12-1639-42), CD45-PE (eBioscience, Cat No. 12-9459-42) at saturating concentrations for 30 min at 4°C and washed twice with FACS medium. Monocyte cell populations (CD45⁺/CMFDA^-) were then identified and gated to measure the expression of cell surface markers.

ADSCs (passages 4–6) were analysed by flow cytometry for cellular membrane expression using CD105-PECy7, CD73-APC, CD90-FITC, CD34-PE, CD14-APC-Cy7 and CD45-PerCP-Cy5 antibodies (all from eBioscience, San Diego, CA).
The capacity of ADSCs to differentiate into osteoblasts and adipocytes was assessed as previously described [19 (link), 20 (link)]. ADSCs were treated with an Adipogenesis Differentiation Kit and an Osteogenesis Differentiation Kit (both from Gibco, Invitrogen Corporation, Carlsbad, CA). The medium was completely changed twice per week. After 3 weeks of differentiation, the ADSCs were stained with Oil Red O and Alizarin Red S. Briefly, ADSCs were washed 3 times with PBS and then fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature. The cells were then stained with 0.5% Oil Red O solution for 60 min at room temperature followed by 0.5% Alizarin Red S (both from Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature. The results were assessed from images captured on an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).

ADSCs (passage 3-6) were analysed via flow cytometry with respect to cellular membrane marker expression using CD105-PECy7, CD73-APC, CD90-FITC, CD34-PE, CD14-APC-Cy7 and CD45-PerCP-Cy5 antibodies (all from eBioscience, San Diego, CA) [43 (link)].
The capacity of ADSCs to differentiate into osteoblasts, adipocytes and chondrocytes was assessed as described [44 (link), 45 (link)]. ADSCs were treated with an Adipogenesis, Osteogenesis and Chondrogenesis Differentiation Kit (Gibco, Invitrogen Corporation, Carlsbad, CA). The medium was changed twice per week. After 3 weeks of differentiation, the ADSCs were stained with Oil Red O and Alizarin Red S. After 4 weeks of differentiation, immunohistochemical staining for type X collagen was performed. The results were recorded using an Olympus FV500 optical microscope (Olympus, Tokyo, Japan).