Fig. 3. LSK cells from naive and immunized mice have equivalent lineage potential.

(A) Sca-1 and c-Kit staining of BM Lin⁻ cells from naive and immunized (2 days after injection of NP₈-CGG/alum) mice. (B) Analysis of LSK cells from naïve (shaded histograms) and immunized (open histograms) mice for FcγRII/III and IL-7Rα expression, compared to expression by GMP (PI⁻Lin⁻c-Kit⁺Sca-1⁻CD34⁺FcγRII/III⁺; broken lines, left histogram) and CLP (PI⁻Lin⁻IL-7Rα⁺c-Kit^lowSca-1^low; broken lines, right histogram), respectively. (C) Erythroid and myeloid potential from single Flt3⁻LSK cells was determined by methylcellulose assay. Flt3⁻LSK cells (100 cells) were sorted from naïve and immunized (d 2) mice and seeded in methylcellulose medium containing SCF, IL-3, IL-6, and erythropoietin. The numbers and types of colonies were determined by optical microscopy 8 d later. The bars show the average numbers of colonies classified as CFU-GEMM (multilineage), CFU-GM (myeloid lineage), and the total number of CFU-G, -M, and -E (single lineage). The standard deviations of the total numbers of colonies are shown. (D) B-cell production by Flt3⁻LSK cells from naïve (●) and immunized (○) mice. Multiple wells of 1, 2, and 8 Flt3⁻LSK cells from naïve and immunized (d 2) mice were cultured on OP9 stromal cell layers in the presence of IL-7 and Flt3L. The frequencies of wells with B-cell growth were determined by the presence of CD45⁺CD11b⁻CD19⁺B220⁺ cells after 14 days in culture.