<p> Cell preparation and cellular characterization by flow cytometry analysis</p><p>Briefly, whole BM from the femurs and tibias was flushed in Basal Medium for</p><p>Murine Mesenchymal Stem Cells supplemented with Mesenchymal Stem Cell</p><p>Stimulatory Supplements (StemCell Technologies Inc., CA), containing 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, UK), by using a 25-G needle. The cell suspension was filtered through a cell strainer (100 μm) to remove debris, followed by centrifugation at 500 g for 5 min. </p><p>After centrifugation, the supernatant was removed. Cells were incubated at a</p><p>7 final concentration of 3 × 10 nucleated cells/ml at 37 °C in a 5% CO2 humidified incubator (SANYO, Japan) for 72 h. Non-adherent cells were removed, and fresh media was added to the adherent cells every 3 to 4 days. When the cells were 80% confluent, adherent cells were digested in 0.05% trypsin (Calbiochem, San Diego,</p><p>CA) at 37 °C for 5 min. Cells were expanded until a homogeneous population was obtained after 2 to 3 weeks of culture, with the population showing a fibroblast-like morphology without any treatment. </p><p>Before further expansion and experimental use, the immunophenotype of</p><p>BMSCs was characterized by CD34, CD45, CD86, CD90, CD106, and MHC-П staining, detected by flow cytometry. Cells were washed with wash buffer (PBS containing 0.09% sodium azide and 2% BSA) and incubated with FITC-conjugated anti-rat-CD34 (eBioscience Inc. USA), CD45, CD90 (Caltag Inc. USA), and CD106</p><p>(eBioscience Inc.), PE-conjugated anti-rat-CD86, and MHC-П (eBioscience Inc.) antibodies in wash buffer for 30 min on ice in the dark. Cells were washed and detected with a flow cytometer. Samples were analyzed within 24 h with BD</p><p>FACScan (BD Biosciences) using Cell Quest software (BD Biosciences). Isotype- matched PE- and FITC-conjugated monoclonal antibodies (mAbs) of irrelevant specificity were tested as negative controls. </p><p>After incubation for 3 days, the CFSCs and nearly all of the BMSCs were attached to the culture flask with different shapes. The CFSCs were polygonal cells</p><p>(Fig. 1A). BMSCs were characterized morphologically by a small cell body, with a few long and thin cell processes. They were expanded until a homogenous population was obtained (about 2–3 weeks). Thereafter, they showed a fibroblast-like morphology without any treatment (Fig. 1B). Before further expansion and experimental use, the cells were characterized according to their expression of CD34,</p><p>CD45, CD86, CD90, CD106, and MHC-П by flow cytometry. The data showed constitutive expression of CD90 and CD106, but not of CD34, CD45, CD86, or</p><p>MHC-П, on the surface of BM-derived cells, implying that these cells were BMSCs. </p><p>Figure 1. Primary cell morphology and molecular expression. (A) CFSCs (200 ×).</p><p>(B) BMSCs were uniform and fibroblast-like (200 ×). (C) FCM analysis showed that from the 3rd generation, BMSCs stably expressed CD106 and CD90, but not CD34,</p><p>CD45, CD86, or MHC-П.</p>