Evaluating the Effects of Cell Sorting on Gene Expression

Abstract

Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison

“…To compare the methodologies of INTACT and FANS for cell-type-specific isolation, we first conducted a literature review to identify and summarize shared and distinct features of both techniques. We decided to limit the range of studies and focus on Mo et al’s [ 10 ] citations, since we based our experimental comparisons on the same reporter line (R26 CAG-Sun1-sfGFP-Myc ). Additionally, this publication reports the first use of INTACT in the mammalian system, providing an essential landmark in the field.…”

“…On the other hand, “ mus musculus ” was the most frequently used organism in the selected studies with 42% of the citations along with “ Homo sapiens ” (12%), “ Arabidopsis thaliana ” (5.8%), and “ Drosophila melanogaster ” (2.3%) ( Figure 1 D, Supplementary Materials Figure S1E,F ). The frequently used tissue and organisms are not surprising, as Mo et al [ 10 ] investigated epigenetic signature changes occurring in mature neurons of transgenic mice.…”

“…To provide a comprehensive comparison between INTACT- and FANS-isolated nuclei, we used the tamoxifen (TAM)-dependent Arc creERT2 (TG/WT) .R26 CAG-Sun1-sfGFP-Myc (M/WT or M/M) mouse line [ 10 , 29 ]. The mouse line expresses sfGFP on the nuclear membrane of specific stimulus-activated cells (expressing Arc) upon TAM injection.…”

“…Additionally, it is challenging to sort morphologically complex cells such as cells from the central nervous system [ 8 , 9 ]. The high hydrodynamic stress forms one of the major challenges [ 10 , 11 ], as this could trigger cellular stress responses leading to a subsequent modification of different molecular profiles [ 8 , 12 , 13 , 14 ]. Apart from this, FACS/FANS also comes with a high time–cost factor to isolate rare cell populations at a multiscale level [ 1 , 9 , 15 ].…”

“…Since then, this method has been adapted to model organisms such as C. elegans, D. melanogaster [ 16 , 17 ], and other mammalian systems [ 18 , 19 ]. Mo and colleagues introduced INTACT to sort activated neuronal (sfGFP+) populations in the mammalian brain [ 10 , 20 ]. Since then, the use of INTACT has been extended to distinct neuronal cells [ 18 , 19 , 21 , 22 ], cardiomyocytes [ 23 , 24 , 25 ], and hepatocytes [ 26 , 27 ].…”