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Blood, Vol. 113, Issue 13, 2914-2923, March 26, 2009
The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice
Blood Yokota et al.
113: 2914
Supplemental materials for: Yokota et al
Files in this Data Supplement:
- Figure S1. A sorting strategy for HSC-enriched and ELP-enriched fractions (JPG, 134 KB)
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The Rag1− c-kitHi Sca1+, HSC-enriched fraction and the Rag1Lo c-kitHi Sca1+, ELP-enriched fractions were isolated from E14.5 liver of Rag1/GFP knock-in mice by 2-step sorting with a FACSaria. In the first step, Rag1− or Rag1Lo cells were sorted according to levels of GFP expression by collecting data in two fluorescence channels without compensation (upper panels). The sorted cells were stained with PE–anti-Sca1 and APC-anti–c-kit antibodies, suspended in 7AAD-containing buffer, and subjected to a second round of sorting (middle and lower panels). Dead cells stained with 7AAD (far left) and a few contaminating cells with inappropriate GFP levels (near left) were gated out, and the cells were then fractionated according to Sca1 and c-kit to obtain Rag1− c-kitHi Sca1+ HSC and Rag1Lo c-kitHi Sca1+ ELP (near right). Purities of the sorted cells were verified by post-sort analyses, in which small aliquots of each sorted fraction were tested immediately after the sorting (far right).
- Figure S2. E9.5 YS hematopoietic cells differ from those in the embryo proper with respect to ESAM expression and lymphopoietic activity (JPG, 89.4 KB)
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YS or the caudal half of embryo proper containing the PSp/AGM region were obtained and pooled from E9.5 embryos of WT C57B6 mice. The tissues were dissociated, and then the obtained cells were stained with the anti-ESAM Ab (1G8) followed by goat anti-rat IgG-FITC, anti–c-kit–APC, anti–Tie2-PE, and 7AAD. (A) The profiles of Tie2 and c-kit expression are shown in the left panels. In the right panels, ESAM expression in each gate is shown in histograms. The sorted fractions were labeled with “A” to “F.” The sorted cells were subjected to methylcellulose colony formation assay (B) and tested in the MS5 co-culture system (C). Cultured cell numbers in methylcellulose were; gate A: 600 cells per dish, gate B: 2300 cells per dish, gate C: 700 cells per dish, gate D: 290 cells per dish, gate E: 960 cells per dish, gate F: 260 cells per dish.
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