RESUMO
Isolation of multiple cell populations from limited starting material and with minimal influence on cell homeostasis and viability are common requirements in both basic and clinical research. Fluorescence-activated cell sorting (FACS) is the most commonly applied sorting methodology with the majority of instruments being based on high pressure and electrostatic deflection. A more recent technology is based on a mechanical valve, operating at low pressure. In the present work we compared the two technologies by parallel sorting of small amounts of peripheral blood and umbilical cord blood on a BD FACSAria™ III and Miltenyi MACSQuant® Tyto® instrument. Concurrent manually performed magnetic-based cell sorting served as reference. Sorting metrics, including purity and viability, were compared. Expression of the heat-shock protein HSPA1A immediately post sorting and the proliferation potential of sorted T-cells in vitro was assessed. In general, there was little to distinguish the two fluorescence-activated technologies with regard to sorting metrics and HSPA1A expression. Variation, however, with respect to recovery and viability, was much smaller among Tyto sorted samples. The proliferation potential of Tyto-sorted T-cells was significantly higher compared to Aria-sorted T-cells, indicating that T-cells of the Tyto instrument are less perturbed. In summary, cell types of blood origin including CD34+ cells could effectively be isolated from small input amounts with either fluorescence-activated technology with little immediate effect on viability. The mechanical valve-based sorting by the Tyto instrument; however, appeared to perturb the cells to a lesser extent as judged by their proliferation potential.
