Microenvironment contamination occurs from sorted HSPCs but does not reflect a stem cell niche–type interaction with stromal cells. Different populations of HSPCs (Lin⁻ Sca-1⁺ Kit⁺ [LSK], Lin⁻ Sca-1⁻ Kit⁺ [LK], Lin⁻ Sca-1⁻ Kit⁻ [L3⁻]) that were FACS purified (purity > 90% for all fractions) were stained with 2μM DiI, washed, and incubated overnight in serum-free medium complemented with cytokines. After washing, cells were plated on confluent layers of endothelial cells (HUVECs) or fibroblastic cells (GP293). After the coculture, dye transfer was analyzed by confocal imaging (B) and FC (A,C). (A) Cells were recovered by trypsinization and stained with anti–mCD45-APC. After washing, cells were suspended in DAPI containing PBS, 2% FCS for FC analysis. After elimination of debris and doublets, viability (DAPI⁻) was determined on HSPCs (CD45⁺). (B) After overnight coculture (18 hours), hematopoietic cells were stained in situ by an anti–mCD45-APC antibody. A 10-μmstack was acquired for each region of interest (2-μm step) using a zoom 3 magnification. Displayed are 3-dimensional reconstructions for individual and merged channels for the cocultures of LSK and LK on endothelial cells. Scale bar represents 20 μm. (C) Absolute quantification of DiI distribution after 22 hours of coculture: using the same strategy as in Figure 2D, absolute quantification of DiI fluorescence was measured for HSPCs (CD45⁺) and stromal cells (CD45⁻) and expressed as percentage of total DiI fluorescence in the different conditions.