Supplementary Figure 7: Interactions of HSCs and nucleated erythroid cells in the BM.

(a) Representative flow cytometry histograms showing staining by antibodies against CD48, Lineage (Lin; B220, CD3, CD11b, Gr1), CD150 and CD71 used for identification of HSCs and nucleated erythroid cells (NECs). The HSCs (LSK CD150⁺ CD48^–) are distinguished as Lin^– CD150⁺CD48^–CD71^–; the NECs (CD71⁺ Ter119⁺) are CD71⁺Lin^–CD150^–CD48^–; the MPCs and CD48⁺LSK subset are Lin^–CD48⁺ CD150^–CD71^– and the lineage cells are Lin⁺CD48⁺. (b) Application of the above combination of antibodies to the two-photon microscopy of the whole-mounted femur. HSCs (Lin^–CD150⁺CD71^–; green) and NECs (Lin^–CD150^– CD71⁺; red) appear clearly distinct. HSCs (in the upper rows of magnification insets, green; in lower rows of insets, only outlined in white) did not stain for lineage or CD71. Scale bars, 50 μm and 40 μm (c) Representative two photon microscopy images of the whole-mounted femurs from ACKR1-deficient mice used for the measurements of the distance between NECs and HSCs. (d) Quantitative analysis of HSC, MMP1, MPP2 and LRP populations interacting with NECs assessed by flow cytometry. Wild-type (WT, blue) and ACKR1-deficient (KO, red). Two-tailed Student’s t-test, (n=3). All numeric data are Mean±SEM. ***P < 0.001.