Immunophenotyping of Bone Marrow Stem Cells

The expression of mesenchymal and non-mesenchymal stem cell-associated surface markers that are the characters of the immunophenotype of BMSCs were measured by flow cytometric analysis at early passages (P3). Briefly, approximately 5 × 10⁵ liberated adherent BMSCs were detached from the culture flasks and suspended in PBS containing 3% FBS in different EP tubes, and the following antibodies were incubated for 20 min at room temperature in the dark: CD4, CD44, CD45, CD90, CD105 (eBioscience, U.S.A.). A negative panel of the following surface antigens was incubated simultaneously as a group in the same sample. Cell suspension without antibodies served as a control group to determine background fluorescence. After 1 h, the cells were washed with PBS containing 3% FBS for three times and 300 ml of the suspension was added into the testing tubes, equipped with laser emission at 488, 633 and 407 nm. The FITC and PE channels were used to detect the emission of conjugated surface antigens [23 (link)]. Finally, the samples were measured by flow cytometric analysis using a Beckman Coulter Epics XL cytometer (Beckman Coulter, Fullerton, CA, U.S.A.).