2.7. PKH-26 Staining

PKH-26 is a cell membrane fluorescent dye and is divided to daughter cells. The distribution of PKH-26 in the daughter cells was tracked, and a mathematic model provided by Cicalese et al. was used to define the division patterns [11]. PKH-26 dye was diluted as indicated and incubated with PBS-washed cells for 5 min at room temperature. The staining reaction was ceased by adding 2 ml serum. The cells were washed and centrifuged to remove unbound excess dye, resuspended in serum-free DMEM medium supplemented with 20 ng/ml EGF, 20 ng/ml bFGF, and 4 μg/ml insulin. Single cells were obtained by serial dilutions in a 96-well plate, labeled, and observed under fluorescence microscopy. The images were acquired by confocal microscopy.