Cd105 pc7

hMSC characterization by flow cytometry was performed on cells before seeding on biomaterials to demonstrate their characteristics as defined by the ISCT criteria (Dominici et al., 2006 (link)). hMSC were stained with CD90-FITC, CD73-PE, CD105-PC7, CD45-APC-A750 antibodies (Beckman Coulter, Brea, California, United States), or corresponding isotype controls in matched concentration, 20 min at room temperature (RT) and in the dark. Cells were washed in PBS without Ca²⁺/Mg²⁺ (Thermo Fisher), fixed, and permeabilized (IntraPrep Permeabilization Kit, Beckman Coulter). Intracellular staining was performed by an indirect immunofluorescence method with an adapted dilution of Nestin antibody (Merck Millipore, Burlington, Massachusetts, United States), or its corresponding isotype control in matched concentration, and with an Alexa Fluor 647 goat anti-mouse IgG (H + L) secondary antibody (Thermo Fisher). After washing, cells were analyzed with a NAVIOS flow cytometer (Beckman Coulter) and data files were interpretated using Kaluza software (Beckman Coulter).

Preadipocytes and DFATc from passage 4 were seeded in a 10-cm petri dish in growth medium until confluence. Cells were then washed with PBS, and trypsinized and resuspended (5 × 10⁶ cells/mL) in staining buffer (PBS, sodium azide 0.02%, and serum bovine albumin 0.2%). Cells (100 μL) were incubated 20 min with propidium iodure and the following anti-human primary antibodies: CD31-PB, CD45-KO, CD90-APC, and CD105-PC7 (all from Beckman Coulter). After incubation, cells were washed twice with PBS, centrifuged 5 min at 150g, and resuspended in staining buffer (0.5 mL). Matrix of compensation was generated using VersaComp Antibodycapture Beads kit (Beckman Coulter), according to the manufacturer’s instructions. Data were acquired using Gallios (Beckman Coulter), and analysis was performed using Kaluza analysis software (Beckman Coulter).