Cell Preparation and Cellular Characterization by Flow Cytometry Analysis

Cell preparation and cellular characterization by flow cytometry analysis

Briefly, whole BM from the femurs and tibias was flushed in Basal Medium for

Murine Mesenchymal Stem Cells supplemented with Mesenchymal Stem Cell

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.

After centrifugation, the supernatant was removed. Cells were incubated at a

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,

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.

Before further expansion and experimental use, the immunophenotype of

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

(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

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.

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

(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,

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

MHC-П, on the surface of BM-derived cells, implying that these cells were BMSCs.

Figure 1. Primary cell morphology and molecular expression. (A) CFSCs (200 ×).

(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,

CD45, CD86, or MHC-П.