Anti cd90 pe

Cell cycle analysis was carried out using propidium iodine (PI) staining. In short, after exposure to ⁹⁰SrCl₂, cells were detached by trypsin incubation, washed twice and counted. 300,000 cells were fixed in 70% ethanol and stored at −20 °C for at least one hour. Cells were then centrifuged 8 minutes at 400 g and the pellet was re-suspended with a sodium citrate solution containing 0.2% Triton X-100, 100 μg.mL⁻¹ RNAse and 50 μg.mL⁻¹ PI (Sigma) and incubated 30 minutes at 37 °C in the dark. Stained cells were then analyzed on a FACS Canto II (BDIS, Le Pont-de-Claix, France) with the acquisition of at least 10,000 events per condition.
Phenotypic analysis of rat BMSCs was performed using the following directly coupled antibodies: anti-IgG1-FITC, anti-IgG2a-PE, anti-CD44-FITC, anti-CD45-FITC, anti-CD90-PE, anti-CD73-alexa647 (all from BD Pharmingen, Le Pont-de-Claix, France) and anti-CD34-PE (Santa Cruz Biotechnologies). At the first passage, aliquots of 200,000 cells were washed twice in PBS and incubated for 20 minutes in the presence of the indicated antibodies at pre-defined concentrations. Cells were then washed twice and analyzed.

Flow cytometry was performed by using an LSRII four-laser flow cytometer (BD Biosciences, Oxford, UK) with appropriate isotype controls. Passaged MSCs (p3 from HN, SF, and ICBM) (n = 3 for each group) were stained with combinations of the following antibodies at the dilution recommended by the manufacturers: anti-CD14-allophycocyanin-cyanine (APC-H7), anti-CD19-fluorescein isothiocyanate (FITC), anti-CD34-peridinin chlorophyll protein (PerCP), anti-HLADR-phycoerythrin-cyanine (PE-Cy7), anti-CD73-phycoerythrin (PE), anti-CD90-PE, anti-CD81-FITC, anti-CD44-FITC, and anti-CD29-FITC (all from BD Biosciences). Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) as a live/dead discriminator immediately prior to acquisition
[15 (link)]. At least 10,000 live cell events were collected for each antibody combination.

Cells were stained with anti-CD31-PE, anti-CD33-FITC, anti-CD34-PE, anti-CD45-FTIC, anti-CD13-PE, anti-CD44-PE, anti-CD45-FITC, anti-CD71-PE, anti-CD90-PE, anti-CD95-APC, anti-HLA-ABC-FITC, anti-HLA-DR-FITC (all from BD Bioscience), anti-CD31-APC (eBioscience), anti-CD13-PE (Biolegend) and anti-CD105-FITC (R&D Systems). The stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).