Fab10971f

Cell surface marker expression was analyzed for cells at passage 4 (P4) as previously described [21 (link)]. The cells were stained for 30 min with FITC-conjugated anti-rat CD105 (R&D, FAB10971F), PE-conjugated anti-rat CD73 (R&D, FAB5796P), PE-conjugated anti-rat CD34 (Beckman coulter, IM3630A), antibodies, and PE-conjugated anti-rat CD14 (Beckman coulter, IM0650U) antibodies (Beckman Coulter, Brea, CA, USA). For gating, unstained cells were used as controls. Expression of exosome surface markers was analyzed for BMSCs-Exo as previously described using PE-conjugated anti-mouse CD63 (BioLegend, San Diego, CA). Analysis was performed using a CYTOMICS FC 500 Flow Cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using CXP Software version 2.2. Additionally, analysis of the exosomes using transmission electron microscopy (TEM) was performed after suspending the exosomes in PBS buffer. This suspension was applied onto a carbon-coated copper grid, followed by staining with 2% uranyl acetate. Images of exosomes were obtained using an electron microscope (JEM-1010; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 70 kV.

Per condition, 2 x 10⁵ MSCs were re-suspended in 500 μl FACSFlow solution (BD Biosciences) and stained with antibodies against human CD45-APC (368515, BioLegend), CD90-APC (FAB2067A, R&D Systems), CD73-PE (550257, BD Biosciences), or CD105-FITC (FAB10971F, R&D Systems), following the manufacturer's guidelines. Afterwards, the cells were fixed using 2% formaldehyde (Fluka) and were filtered through 70-μM filters. Unstained cells were used as a negative control. Samples were analyzed by flow cytometry using a BD Fortessa machine (BD Biosciences). The data were analyzed using FlowJo V10 software.