CD86 as an alternative marker for Sca-1. (A) Expression of CD86 on LKs and LSKs at steady state. (B) Expression of CD86 on LKs, LT-HSCs, short-term HSCs (ST-HSC), MPP2, and MPP3/MMP4 in naive WT mice was examined by FCM. (C) Expression of Sca-1 on L86Ks and CD86⁻ LKs before and 24 hours after the LPS injection. (D) Kinetic analysis of cell numbers for LSKs, L86Ks, Sca-1⁻ LKs, and CD86⁻ LKs before and 6 and 18 hours after LPS treatment; n = 3 per group. (E) Evaluation of Sca-1 expression in CD86⁻ LKs or L86Ks in WT and Stat1^−/− mice 18 hours after LPS treatment. Sca-1 expression of CD86⁻ LKs (blue) and L86Ks (red) in the left panels are shown in the right panels. (F) Evaluation of Sca-1 and CD86 upregulation by an ex vivo culture system. Lin⁻c-kit⁺Sca-1⁻CD86⁻ BM cells were isolated from naive CD45.2⁺ mice and cocultured with total BM cells (CD45.1⁺) in the absence or presence of LPS (100 ng/mL) for 12 hours. Statistical evaluation is shown in the right panel. (G) Evaluation of CD86 upregulation in vivo. Lin⁻c-kit⁺Sca-1⁻CD86⁻ BM cells isolated from the BM of naive CD45.2⁺ mice were directly injected into the BM of CD45.1⁺ mice. Twelve hours after LPS injection, the expression of Sca-1 and CD86 on CD45.2⁺ donor cells was examined. Statistical evaluation is shown in the right panel. (H) t-SNE analysis for LSK and L86Ks before and 24 hours after LPS injection. LSKs and L86Ks were divided into 3 groups (blue, red, and green) based on the distribution on t-SNE plots (i). The expression patterns of CD34 and FcγR (ii) or Sca-1 (iii) were assessed. (I) Expression of Flt3 on MEPs, CMPs, and GMPs in naive conditions. (J) Frequencies of Flt3⁺CD34⁺ cells in LKs, LSKs, and/or L86Ks before and/or 24 hours after LPS treatment. The numbers on FCM plots indicate frequencies of gated populations. *P < .05, 1-way ANOVA (F and G). Data are representative of 2 (E-J) or 3 (A-D) independent experiments (error bars in panels D, F, and G represent SEM).