Cytometry of Reaction Rate Constant (Crrc): Optimization of the Technique and Development of a Novel Protocol to Assess Cell Population Heterogeneity Based on Aldh1a1 Activity in Vitro

Cytometry of Reaction Rate Constant (CRRC) is a novel analytical technique which aims to study cellular heterogeneity based on the activity of enzymatic reactions. In the past, CRRC was able to assess cellular heterogeneity in vitro by investigating the activity of ABC-transporter enzymes. However, CRRC showed poor robustness to highly motile cells. Here, I report on the development of a workflow to make CRRC robust to cell motility. The novel workflow was used to develop a protocol for in vitro CRRC studies of aldehyde dehydrogenase 1A1 (ALDH1A1)-based cell heterogeneity. The data collected suggested a potential positive correlation between the activity of ALDH1A1 and the age of a cell line. Finally, I demonstrated the robustness of the new CRRC ALDH1A1 assay to a 20% change in the initial substrate concentration. Overall, these studies confirm the potential for CRRC to become a reliable analytical tool for studies of reaction-based cell heterogeneity.