Slowly dividing cells divide asymmetrically. (a, b) 4T1 cells were labeled with Vybrant CM-DiI (red fluorescence) and then single cells were seeded into individual wells of 48-well plates (one cell/well). (Panel a) Forty-six out of 48 wells showed homogenous distribution of Vybrant CM-DiI to the daughter cells (rapidly dividing cells). (Panel b) Two out of 48 wells showed a few highly labeled cells (slowly dividing cells), whereas the rest of the colony became gradually unlabeled (rapidly dividing cells). This heterogeneous distribution of Vybrant CM-DiI suggests asymmetric cell division of slowly dividing cells giving rise to one daughter cell that stays slowly dividing and retains the label (self-renewing cell), whereas the other daughter cell converts into a rapidly dividing cell. (c, d) Colonies as shown in panels a and b were dissociated, labeled with DiO (green fluorescence), and again seeded as single cells (one cell/well). (Panel c) Colonies formed from rapidly dividing cells (no red fluorescence) continued to divide rapidly and showed homogenous distribution of DiO until the fluorescence signal was diluted below the threshold of detection. (Panel d) Colonies formed from slowly dividing cells (red fluorescence from Vybrant CM-DiI labeling was still visible) again showed asymmetric cell division giving rise to slowly dividing cells that were labeled with both Vybrant CM-DiI and DiO (arrow), and rapidly dividing cells that became unlabeled. A schematic representation is provided in the additional materials [see Figure S2 in Additional data file 1]. Scale bars = 5 μm.