Figure 2

In situ fluorescent staining can be used to measure dose-dependent effects of TGFβ1 on mature ECM accumulation. (A) Human RPTEC were stimulated with different concentrations of TGFβ1. The decellularised matrix was fixed and stained in situ with Flamingo fluorescent dye. Images are representative of a single field. (B) High-content analysis of the data collected in A. was performed using the Cellomics “Cell Health Profiling” bioapplication to obtain fluorescence intensity data for decellularised accumulated mature ECM stained in situ with Flamingo fluorescent dye. The PrestoBlue signal was used to normalise for differences in cell number. The dose-response curve shows mean target fluorescence intensity normalised to the PrestoBlue signal (mean ± SD of 6 replicates). (C) Incorporation of ¹⁴C-labelled amino acids into the deposited ECM was measured following stimulation of human RPTEC with differing concentrations of TGFβ1. The dose-response curve shows the radioactivity count for the ECM normalised to the total cell count for the radioactive endpoint (mean ± SD of 8 replicates). Figures (B) and (C) show representative curves from at least 3 independent experiments and data from the individual repeats is summarised in Table 1.