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A. O’Neill et al.
Ly6C/G and propidium iodide was added to identify live cells. Lineages were gated as shown in Online Supplementary Figure S1 and colonies were defined by the presence of >20 cells of any one of each lineage.
Cell cycle analysis
cKit-enriched BMMNC were isolated as above and stained with PerCP-Cy5.5 lineage cocktail, FITC-conjugated CD34, PE- conjugated CD41, PE-Cy7-conjugated Sca1, APC-conjugated CD150 and APC-Cy7-conjugated cKit. Cells were then fixed and permeabilized by IntraPrep Reagent (Beckman Coulter) in accor- dance with the manufacturer’s protocol. AlexaFluor700-conju- gated Ki67 (SolA15, Invitrogen) antibodies and Hoechst 33342
were added after permeabilization and cells were analyzed by flow cytometry.
Rescue experiments
For the rescue experiments, 1 mg CXCL4/PF4 (Peprotech) or PBS was injected intraperitoneally daily for 7 days and bone marrow analyzed on day 7. Romiplostim (Kyowa Kirin) or PBS as a control was administered intravenously at a dose of 100 mg/kg per day for 5 days.
Statistical analysis
All values given are mean ± standard deviation. Statistical analyses were performed by two-tailed unpaired t-tests and the
AB
CD
EF
GHI
Figure 3. Hematopoietic stem and pro- genitor cells show variable responses to loss of thrombopoietin/MPL signal- ing. (A) Flow cytometric gating system for hematopoietic stem and progenitor cell (HSPC) populations. (B-D) Cell counts of populations in bone marrow of ThpoΔ/Δ Alb-Cre, Thpofl/fl, Thpo-/- and ThpoWT/WT mice were analyzed for long- term hematopoietic stem cells (LT-HSC) (B), CD150- HSPC (C) and CD41+ HSPC (D) (n=6). (E) A representative plot of CD48 and MPL expression in LSK cells, with CD48 and MPL gating lines indicat- ed. (F) Percentage of wild-type (WT) cells from each HSPC population that are CD48- (n=8). (G) Percentage of CD48- cells from each population expressing MPL (n=8). (H) MPL mean fluorescence intensity (MFI) of CD48- MPL+ cells from each population (n=8). (I) MPL mRNA expression levels relative to GAPDH (n=3). BMMNC: bone marrow mononuclear cells.
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haematologica | 2021; 106(7)