Addition of low concentrations of imatinib (50 nM) to the media maximally increased the number of CFU-GM by 42% compared to untreated marrow (Fig. flow cytometry contour plots gated on total live singlet splenocytes from a control animal, an animal treated with imatinib (66mg/kg/d), a Mm-infected animal, and an infected animal treated with imatinib. The box on the right of each panel represents neutrophils. The total number of cells, and the number of neutrophils is listed below each plot, and the percentage of neutrophils relative to total live cells is shown within the box (C) Effects of imatinib on DC cell subtypes. Beginning 24h post-treatment mice were either injected in the tail vein with 105 CFU Mm 1218R or left uninfected. CD8+ DC (CD11chi CD8+), CD8- DC (CD11chi CD11b+ CD8?) and pDC (CD11clo CD11b? B220+ and Ly6C+) numbers were enumerated by flow cytometry in the blood (top panel) or spleen (bottom panel) at d7 post infection or treatment. Combined data from NPB two to three independent experiments are presented with 6 mice per condition. A Mann-Whitney test was used for pairwise comparisons, and a Kruskal Wallis test for multiple comparisons.(TIF) ppat.1004770.s001.tif (1.3M) GUID:?48861457-5E83-412F-884F-04E013AB0FC1 S2 Fig: Gating schemes for isolation of mature myeloid and lymphoid cells, myeloid precursors and progenitors in bone marrow. (A) Gating scheme for isolation of mature myeloid and lymphoid cells from bone marrow in Fig. 2. (B) DC subsets from bone marrow of imatinib-treated or infected mice. C57Bl/6 mice were administered imatinib at 66 mg/kg/d or left untreated. Beginning 24h after onset of drug, mice were either injected in the tail vein with 105 CFU Mm 1218R or left uninfected. At 7 days post-treatment bone marrow was collected from femurs. CD8+, CD8- and pDCs were enumerated by flow cytometry. The line in each data set represents the median. A Mann-Whitney test was used for pairwise comparisons, and a Kruskal-Wallis test for multiple comparisons. Combined data from two independent experiments are shown.(TIF) ppat.1004770.s002.tif (1.0M) GUID:?02BFBCA5-7BC1-4829-B732-0B478599D345 S3 Fig: Gating scheme for isolation of myeloid precursors in bone marrow. (A) Representative flow cytometry contour plot of bone marrow subset gating based on CD45 expression and side scatter (SSC): lymphocytes (yellow), monocytes (green), promyelocytes (orange), myelocytes/metamyelocytes (pink), mature neutrophils (red), and total granulocytes including all neutrophil progenitor NPB populations and NPB mature neutrophils (blue). (B) Frequency of total live bone marrow cells of subsets. Combined data from two independent experiments are presented with n = 6 mice per condition. Bar in each data set represents the median+/-SEM. A Mann-Whitney nonparametric test was used to determine significance.(TIF) ppat.1004770.s003.tif (534K) GUID:?F88C1404-F693-4EEE-9F5B-EDCF987BD3B2 Gdnf S4 Fig: Gating scheme for isolation of hematopoietic stem cells, and myeloid progenitors in bone marrow. (A) Gating Scheme for isolation of LSK, HSC, MPP1, MPP2, MPP3 and MPP4 cells from bone marrow (adapted from ). Following selection of the lineage negative population, cells were phenotyped for expression of c-Kit and Sca-1 to select the LSK cells (lineage?, c-Kit+ and Sca-1+). LSK cells were then divided into populations A, B and C according to their expression of CD48 and CD150. The co-expression of CD34 and CD135 were then used to delineate the HSC and MPP subpopulations (MPP1-4). (B) Representative plots of LSK cells with or without imatinib. Plots show selection of linage negative cells phenotyped with c-Kit and Sca-1. LSK cells, which are Sca-1+ and c-Kit+, are NPB delineated by the boxes with the overall percentage indicated. The panels are representative, and derived from a control animal (left panel), or and animal treated with imatinib (66mg/kg/d) for 7d (right panel). (C) Effects of ACK2 antibody during infection with Mm. ACK2 or 2A3 (isotype control) (10mg) was injected intravenously every 48h for six days prior to infection with Mm. CFU were measured 48 hours later. Data shown are from a.