Figure 2.

Detection of ALDH activity and ALDH^highCD44⁺ cells within the ALDH^high and ALDH^low fractions of the three MPM cell lines. (A) Representative FACS analysis of ALDH activity in H28, H2052 and Meso4 using Aldefluor assay. Baseline control of ALDH fluorescence was established by the addition of ALDH inhibitor, DEAB (+DEAB) and used to provide the ALDH^high region for cells without DEAB (- DEAB). The same gating principle was applied to sort for ALDH^high cells. (B) Representative FACS-based sorting of ALDH^high and ALDH^low fractions for H2052. The non-viable cells (PI⁺ cells) were excluded before gating the ALDH^high cells. In all cases, ALDH^low cells represent <4% of the dimmest cells relative to the analysed population. Average purity of ALDH^high cells was ≥98%. (C) The freshly-sorted ALDH^high and ALDH^low fractions were immediately re-stained with CD44 APC-H7 antibody to determine the co-expression of ALDH and CD44. The same procedures were employed for ALDH sorting of H28 and H2052 cell lines and to determine the co-expression of ALDH and CD44. Three independent experiments were done for each cell line.