Quantitative imaging analysis of VEGF mutant ES cell cultures.

Day 8 ES cells were processed as shown in Figure 1, and imaging analysis was performed as described in “Materials and methods.” (A) Percentage area stained with antibody to ICAM-2 in 2 separate experiments. W, wild-type (+/+) VEGF cultures; H, hemizygous (+/−) VEGF cultures; N, null (−/−) VEGF cultures. In all cases, (+) after the letter denotes the same genotype ES cells incubated with 15 ng/mL recombinant hVEGF-165 from days 3 to 8. (B) Percentage area stained with antibody to PECAM or ICAM-2 in duplicate wells of the same experiment. Abbreviations are as for A. In all cases, statistical analysis was performed comparing H and N figures to the W figure in the same experiment, and each (+) figure was compared to the nonrescued figure for the same genotype. *P < .01; **P < .001. (C) Semiquantitative assessment of PECAM+ aggregates (examples in Figures 1F, 1G) was conducted by visual inspection of 2 to 4 wells of each genotype in separate experiments. The relative abundance of the aggregate staining was estimated as follows: +/−, trace; 1+, 2% to 5%; 2+, 5% to 10%; 3+, 10% to 30%, 4+, more than 30%.