Proliferation Analysis of Subconfluent Stromal Cells

The proliferative activity of subconfluent sl-pHCs was determined with regard to distribution in the cell cycle and population doubling time (PDT). The cells were stained with Muse® Cell Cycle Kit and Muse® Count and Viability Kit, respectively. The analysis was performed on Muse Cell Analyzer – the reagents and analyser derived from Luminex/Merck (Poznan, Poland). The number of cells was determined routinely, and obtained data were used to calculate PDT with an algorithm published by Heuer et al. [11 (link)] and Cell Calculator [12 ]. The proliferation of sl-pHCs was also evaluated based on the BrdU incorporation assay (Abcam, Symbios, Straszyn, Polska). For BrdU analysis, cells were seeded at density 2.5 × 10³on a 96-well plate. All tests were performed accordingly to well-described methods [8 (link), 13 (link)] and in compliance with protocols provided by the manufacturers of the assays. Furthermore, colony-forming unit (CFU-E) occurrence efficiency was assessed after ten days of culture using the earlier protocol for stromal [8 (link)] and cancer cells [14 (link)].