Cd44 pe

Stem cells were seeded in 25 cm² culture flasks and were harvested by trypsinization after 7 days. Cells were incubated for at least 1 h at room temperature in PBS with 2% FCS to allow re-expression of receptor proteins at the cell surface. For intracellular staining of vimentin (1:100, Millipore) and nestin (1:100, Dako), cells were first fixed and then permeabilized with the cytofix/cytoperm kit (Becton–Dickinson, San Jose, CA, United States) according to manufacturer’s protocol. 0.5 × 10⁵ cells were washed with PBS containing 2% FCS and were incubated for 30 min at room temperature with primary antibodies against either CD24-PE (1:20, eBioscience, San Diego, CA, United States), CD31-PE (1:100, ImmunoTools), CD34-PE (1:100, ImmunoTools), CD44-PE (1:100, Abcam), CD45-PE (1:100, eBioscience), CD90-PE (1:100, eBioscience), CD105-PE (1:100, Abcam), and p75-PE (1:100, Dako). As a negative control for non-specific background staining, appropriate isotype controls were included. Thereafter, cells were washed three times with PBS and if necessary, incubated with secondary antibodies including FITC-labeled goat anti-rabbit (eBioscience) or PE-labeled anti-mouse IgG (Invitrogen) for 45 min at room temperature. Samples were analyzed on a FACScalibur^TM flow cytometer equipped with CellQuest software (BD Biosciences, Erembodegem, Belgium).