Additional Supporting Information may be found in the online version of this article.

CYTO_22093_sm_SuppFig1.jpg3035KSupplemental Figure 1: Comparison of SPKLS profiles on different flow cytometers, demonstrating how key parameters used for hematopoietic stem cell identification and isolation by flow cytometry appears visually on different machines. No FSC / SSC gate is needed as dead cells and erythrocytes are excluded by the SP gate. Hoechst Red and Hoechst Blue parameters are examined first, and the voltages adjusted to place the majority of the cells in the upper right quadrant, allowing the SP cells to be central to the plot. The Hoechst-red parameter also reveals propidium iodide staining, so dead cells can be excluded on this plot (they line up against the right side), as well as red blood cells (no Hoechst stain, so lower left corner), by drawing a rectangular gate. The SP can then be identified, as gated. In normal mouse bone marrow, an appropriately stained and gated SP will comprise around 0.01 to 0.3% of the live cell gate. The SP cells are then displayed for their lineage-marker profile in a histogram (the lineages markers being a cocktail of lineage-specific antibodies). The lineage-negative cells (usually around 85% of the SP) are then gated to a Sca-1 versus c-Kit plot with the double-positive population here taken to finally identify HSCs with the phenotype SP+ / Lineage- / Sca-1+ / c-Kit+ or SPKLS. If the SP has only a much lower percentage of Lineage-c-Kit+Sca-1+ cells than shown here, the Hoechst staining is likely to be poor.
CYTO_22093_sm_SuppFig2.tif1141KSupplemental Figure 2: SPKLS cells from 4 month and 24 moth old mice showed heterogeneous CD150 staining. The SPKLS CD150+ (red) and CD150- (green) cells were backgated to the SP plot. The CD150+ cells were localized to the “SP tip” and the CD150- cells were localized to the “SP top”.
CYTO_22093_sm_SuppTab1.doc101KSupporting Information Table 1

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