Journal: Cell Reports

Article Title: Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches

doi: 10.1016/j.celrep.2021.109618

Figure Lengend Snippet: Responses of the stromal-vascular BM compartment to increased VEGF (A) Flow cytometry analysis of ROS levels by conversion of the probe dichlorodihydrofluorescein diacetate (DCFDA) to the highly fluorescent 2′,7′-di-chlorofluorescein (DCF), in (left) ECs (CD45 − Ter119 − CD31 + ) and (right) hematopoietic cells (CD45 + and/or Ter119 + ) from E16.5 bones (n = 5–7). (B) DCF levels representing ROS in ECs of (left) arteriolar (CD45 − Ter119 − CD31 + Sca-1 + ) and (right) sinusoidal (CD45 − Ter119 − CD31 + Sca-1 − ) subtypes, derived from P2 bones (n = 5–7). (C) MIP (100-μm z axis) of CD31 and Emcn signals in E18.5 tibia BM cavities (dotted lines). Left, overviews; right, metaphyseal (top) and diaphyseal (bottom) regions. (D) Directionality of blood vessels quantified from segmented and skeletonized CD31 staining. Angle 0° = vessel running along the longitudinal bone axis; 90° = parallel to the growth plate zones. Right, area under the curve (AUC) for vessels with directionality between −45° and 45° (n = 4). (E and F) Number of blood vessels per mm 2 (E) and distribution of vessels according to width (F), quantified on segmented CD31 staining images (n = 4). (G) MIP (60-μm depth) of CD31 and Emcn stained tibias at P2. (H) EC counts by flow cytometry (n = 4–8). (I) IHC for phospho-histone H3 (PHH3) and Emcn at P2, and Emcn + PHH3 + mitotic ECs quantified in the dashed areas of the metaphysis and diaphysis (n = 3). (J) Proportions sinusoidal (CD31 + Sca-1 − ) and arteriolar (CD31 + Sca-1 + ) ECs (n = 4–8). (K) MIP spanning 100-μm tissue depth of CD31, Emcn, and Osx-Cre:GFP signals on E18.5 tibias. Also see . (L) Nearest-neighbor distance analysis showing for distinct Osx-Cre:GFP + cells the distance to the closest CD31 + or Emcn + signal. Plots display individual values, median, and interquartile range (no statistics applied). (M) Proportion of triple-negative (TN; CD45 − Ter119 − CD31 − ) CD51 + Sca-1 − and CD51 + Sca-1 + osteolineage progenitors in E18.5 limbs (n = 4–5). (N) TN PαS (PDGFRα + Sca-1 + ) and PDGFRβ + cell numbers in (left) E18.5 and (right) P2 bones (n = 4–7). (O) MIP (20-μm depth) of PDGFRβ, Emcn, and Hoechst signals in (top) E18.5 and (bottom) P2 tibias. (P) TEM of E16.5 tibias. Dotted lines, blood vessels; asterisks, lumens; orange arrows, irregular ruffled membranes; blue arrowheads, pinocytotic vesicles; green block arrows, mitochondria; red arrowheads, fenestrations/pores between zones of tight junctions (#). Graphs, mean ± SEM, except in (L). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, t test. MFI, mean fluorescence intensity. See also and and .

Article Snippet: Detection of reactive oxygen species (ROS) in cells was done using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) (Thermo Fisher Scientific).

Techniques: Flow Cytometry, Derivative Assay, Staining, Blocking Assay, Fluorescence