Sca-1 upregulation skews the apparent numbers and functions of hematopoietic progenitors. (A) Time-course analysis for FCM of Lin⁻ BM cells after LPS treatment. (B) Number and frequencies of LSKs after LPS treatment; n = 3 per time-point. hpi, hours postinjection. (C) Ex vivo cultures of LKs and LSKs under LPS stimulation. LKs and LSKs were sorted from naive CD45.1⁺ and CD45.1⁺/CD45.2⁺ mice, respectively. These cells were mixed and cultured with c-kit⁻ BM cells (CD45.2⁺) in the absence or presence of LPS (100 ng/mL) for 12 hours, and Sca-1 upregulation was assessed by FCM. (D) Evaluation of Sca-1 upregulation on LKs in vivo. LKs obtained from naive CD45.1⁺ mice were directly injected into the BM of CD45.2⁺ mice, and then LPS (5 mg/kg) was injected into the mice. Twelve hours after LPS injection, Sca-1 expression on CD45.1⁺ donor cells was examined in nontreated (n = 5) and LPS-injected (n = 3) mice. IBI, intra-BM injection. (E-F) Tracing of CX3CR1⁺ LKs in vivo. 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, the frequencies of tdTomato⁺ cells in LKs and LSKs were examined. Representative FCM plots and statistical analysis are shown in panels E and F, respectively; n = 3 per group. (G-H) MEPs and LSKs obtained from mice before or 24 hours after the injection of LPS (5 mg/kg) were cultured in methylcellulose medium for 10 days, and cell expansion and TER119⁺ cell generation were evaluated by FCM. Representative FCM plots and statistical analysis are shown in panels G and H, respectively; n = 3 per group. i.p., intraperitoneal. (I-J) MEPs and LSKs isolated from the BM of naïve CD45.1⁺ mice were stimulated with LPS (100 ng/mL) as shown in panel I. Cultured CD45.1⁺ cells were sorted and cultured in methylcellulose medium for 10 days, and cell expansion and TER119⁺ cell generation were assessed by FCM; n = 3 per group. (K-L) Increase in population sizes for LSKs in WT and Stat1^−/− mice before and 18 hours after the LPS injection (5 mg/kg); n = 3 per time-point. Representative FCM plots are shown in (K). (M-N) Evaluation of capacities in LSKs from Stat1^−/− mice. MEPs and/or LSKs were isolated from the BM of WT or in Stat1^−/− mice before and/or after the injection of LPS (5 mg/kg). Cells were cultured in methylcellulose medium for 10 days and the capacities to expand and generate TER119⁺ cells were examined by FCM; n = 3 per group. The numbers on FCM plots indicate frequencies of gated populations. *P < .05, N.S., not significant; Student t test (D) or 1-way ANOVA (F, H, J, and N). Data are representative of 2 (C-F and J-N) or 3 (A, B, G, and H) independent experiments (error bars in panels B, D, F, H, J, L, and M represent SEM).