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Blood, Vol. 113, Issue 26, 6726-6736, June 25, 2009
CEACAM1+ myeloid cells control angiogenesis in inflammation
Blood Horst et al.
113: 6726
Supplemental materials for: Horst et al
Supplemental materials and methods Electron microscopy analyses
Electron microscopy analyses were performed as described.13 Quantification of tube-like structures in the ex vivo top-Matrigel assay
The relative number of junctions in arborizing structures was counted per well in three different experiments to reflect complexity of tube-like networks in this assay. Cells from 3 individuals per experiment and mouse line were used. Tube formation was evaluated from 24 to 120 hrs. Subsequently, data were pooled from these three experiments. A junction was defined as an origin of three or more tube-like protrusions. A schematic drawing (Fig. S3) visualizes the method of quantification used in Fig. S2 at two different time points. The quantification methods used in Figs. 6G and S2 are compared in Fig. S3.
Files in this Data Supplement:
- Figure S1. Histological and electron microscopy analyses of cross sections of Matrigel implants (JPG, 127 KB)
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(A) and (B) show H&E staining in cross sections of Leishmania-containing Matrigel implants derived from B6.WT (A) and B6.Ceacam1−∕− mice (B), ×400. (C) through (F) show transmission electron microscopy analyses of Matrigel implants containing VEGF-C in B6.WT (C) and B6.Ceacam1−∕− mice (D), and (E) and (F) depict analyses from implants that also contained L. major parasites in B6.WT (E) and B6.Ceacam1−∕− mice (F) , all ×1950. H&E stainings as well as electron microscopy studies were performed on 6 specimens each. Here, representative photographs are shown.
- Figure S2. Evaluation of tube-forming capacities in a cell number and time-dependent manner in the ex vivo top-Matrigel assay (JPG, 127 KB)
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(A) and (B) show photographs of the tube-forming assays 24, 48, 72 and 120 hrs post seeding with cells retrieved from Matrigel implants in B6.WT (A) B6.Ceacam1−∕− mice (B), ×200. 5 × 105 cells and 1 × 105 cells were used per experiment, as indicated. The relative number of junctions in arborizing structures was counted per well in three repetitive experiments with 3 individuals per experiment. Comparative quantitative analyses are given in (C) through (F). Data are presented as mean ± SEM, and the numbers of junctions are compared with different amounts of cells used and documented at different time points, as indicated; *P<0.05. Results from cells retrieved from B6.WT (black squares) and B6.Ceacam1−∕− mice (white circles) are shown with each symbol representing the mean of three repetitive experiments with cells from 6 implants in each experiment.
- Figure S3. Schematic representation and comparison of quantification methods used for the ex vivo Matrigel tube-forming assay (JPG, 43.8 KB)
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(A) depicts arborizing structures and their quantification; n=2. (B) Quantification of junctions in tubular networks 24 and 48 hours (n=4 and n=2, respectively) post seeding on top of Matrigel. A junction resembles the origin of three or more branches. Due to network remodelling, total numbers of junctions decrease over time. The asterisks mark individual entities counted for quantification.
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