Does FACS perturb gene expression?

Abstract

Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub-populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short-term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNA's and rapid decreases in their predicted target transcripts. V C 2015 International Society for Advancement of Cytometry Key terms FACS; mammary epithelial cells; miRNA MURINE mammary glands are highly branched tubular organs that develop mostly postnatally, within subcutaneous fat pads. The ducts that comprise the mammary gland arise from multipotent stem cells that generate all of the lineages found in the mature ductal tree (1,2). Differentiated mammary epithelial cells (MECs) can be broadly classified into two populations: luminal epithelial cells that line the ducts, and myoepithelial cells that form a layer surrounding the luminal cells. However, there are multiple cell types within each of these two populations; for example some luminal cells express the estrogen receptor and some do not.To identify the mechanisms that regulate lineage commitment, researchers have conducted transcriptional analysis of discrete mammary cell populations isolated by fluorescence activated cell sorting (FACS) (3,4). These studies all rely on the assumption that robust differences in gene expression between separate lineages are due to endogenous differences in gene expression. However, to our knowledge, this assumption has never been tested. Our study now demonstrates that FACS produces minimal short-term transcriptional artifacts, but also emphasizes the need for care in the methods used to isolate primary cells. MATERIALS AND METHODS MiceA 8-9 week old C3H mice were ordered from Harlan laboratories. Our handling and use of these mice strictly adhered to ACUC-approved protocol (M/12/080). Cell Preparation and RNA ExtractionMammary tissue from the 3rd, 4th, and 5th mammary fat pads was collected (minus the lymph nodes). Each replicate consisted of pooled tissue from three mice. Each mock sorting experiment contained three biological replicates from nine mice.

An Evaluation of T‐Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation

“…The biological status of cells, their viability, vitality, and functionality after a sort is crucial for the validity and outcome of subsequent experiments and has to be evaluated. So far published work (12,26) on instrumentinduced changes use either harsh cell preparation processes or reporter proteins in immortalized cell culture, so that both systems suffered under pre-experimental cellular alterations. In this study, we specifically focused on the identification of cell changes caused by the sorting process, highlighting mechanical and physical forces potentially detrimental.…”

“…osmolarity (7)(8)(9), changes pH, or adds traces of mitogens (10). Additionally, solid tissues or blood derived material require cell homogenization (11), collagenase digestion (12), or the use of anticoagulants like heparin (13); all of them expose cells to considerable stress. Furthermore, certain antibodies alter cell physiology and function by receptor stimulation, blockade (14)(15)(16)(17), or through initiation of cell-or complement-mediated cell lysis (18).…”

Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo

“…However, this method requires high-level expertise, expensive equipment, and is limited by extremely low throughput. Cell sorting can also be used to isolate certain types of cells if a unique cell surface marker is known, or a fluorescent marker can be activated using a Cre-lox dependent method followed by tissue dissociation and fluorescence-activated cell sorting (FACS); this approach can be limited as the dissociation procedure itself can often alter cellular state (Richardson et al, 2015). …”

The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

“…A third and perhaps more obvious challenge is to ensure that the transcriptomes of cells are not significantly altered by the cell isolation process. Although there have been concerns that FACS can perturb gene expression, published validation studies in non‐bone tissues show minimal effects on gene expression with optimized protocols . As also discussed above, cell isolation–induced biases or artifacts can be particularly difficult to exclude when the goal of the experiment is to characterize the effects of environmental changes (such as dietary intake or mechanical loading) in the absence of an internal control (such as a genetic mutation blocking this response).…”