A Subtractive FACS Method for Isolation of Planarian Stem Cells and Neural Cells

Planarians are among the metazoan organisms with the greatest regenerative abilities. This ability is based on their pluripotent stem cells, called neoblasts, which constitute 10-20% of the cells in their body. Elucidating the molecular mechanisms of the planarian stem cell system, for example, the maintenance of stem cell homeostasis and orchestration of lineage choices, contributes powerfully to the advancement of regenerative biology. Our group has developed fluorescence activated cell sorting (FACS) methodologies for the reliable isolation of planarian stem cells, which constitutes an important experimental asset in the field. Here, we describe detailed protocols for the isolation of (1) planarian stem cells and (2) neural cells. Planarian stem cells are isolated by subtraction of the FACS profiles of intact and γ-ray-irradiated (= stem cell depleted) animals stained with Hoechst 33342 and Calcein AM. The neural cells are isolated by subtracting the FACS profiles of head and tail fragments stained with Hoechst 33258 and Merocyanine 540.