Abstract
Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term “sorter induced cell stress” (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted “CD4+” T-cells and “live” cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 μm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed.
Highlights
A wide range of recent findings in biomedical research and clinical investigations are based on data obtained from specific cell populations purified by fluorescence activated cell sorting
Available studies indicate that cell sorting can alter the expression of certain genes in isolated cell pop ulations from mouse mammary glands (Richardson et al, 2015) and in sorted human leukocyte subsets (Beliakova-Bethell et al, 2014)
On a poster presented at the 2014 Association of Biomedical Resource Facil ities (ABRF) Jurkat cells were reported to suffer loss of cellular integrity and displayed altered gene expression a few hours after having been sorted at intentionally harsh conditions
Summary
A wide range of recent findings in biomedical research and clinical investigations are based on data obtained from specific cell populations purified by fluorescence activated cell sorting. Most current conventional cell sorters are droplet producing systems, either “stream-in-air” or “cuvette” based These instruments use pressurized sheath fluid, which passes through a narrow opening (nozzle) to generate droplets at high frequency with the help of an acoustic wave induced by an electric transducer. Sorted cells can be used for various downstream applications, ranging from nucleic acid extractions for molecular investigations to various cell culture-based assays. Due to these principles, fluorescently activated cell sorting has become a fast, accurate, and reliable method to analyze and isolate a great number of different cell types and cell sorting facilities often represent basic infrastructure in biomedical research in stitutions around the globe
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