CD86⁺ LSKs do not show a LK signature. (A) Ratio between CD86⁻ and CD86⁺ cells in LSKs before and 24 hours after LPS treatment; n = 3 per group. (B-C) MEPs and LSKs were isolated from naive mice, and LSKs, CD86⁻ LSKs, and CD86⁺ LSKs were isolated from mice 24 hours after the LPS injection. These cells were cultured in methylcellulose medium for 10 days, and cell expansion and TER119⁺-cell generation were assessed by FCM. Statistical analyses are shown in panel B; n = 6 per group. (D) Gata1 mRNA expression in LKs, LSKs, CD86⁻ LSKs, and CD86⁺ LSKs before and after LPS treatment. Cells were isolated from the BM of WT mice before and/or 24 hours after LPS treatment, and gene expression was evaluated by real-time polymerase chain reaction (PCR); n = 4 per group. (E) Tracing Cx3cr1-expressing cells after LPS treatment. Two days after tamoxifen treatment (2 mg/mouse) in Cx3cr1-CreER/tdTom mice, LPS (5 mg/ kg) or PBS was injected as shown in supplemental Figure 1E. Twelve hours after LPS treatment, frequencies of Sca-1⁺ or CD86⁺ cells in tdTomato⁺ cells were examined. Representative FCM plots of Lin⁻c-kit⁺ BM cells and statistical analysis are shown in the left and right panels, respectively; n = 3 per group. The numbers on FCM plots indicate the frequencies of the gated populations. N.S., not significant. P > .05; *P < .05, χ² test (A) or 1-way ANOVA (C-E). Data are representative of 2 independent experiments (A-C) or 3 independent experiments (D and E) (error bars in panels A, C, and E represent SEM).