Sorting cells alters their redox state and cellular metabolome

Abstract

A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

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

“…Yet, in contrast to previous reports (44,45), the cellular sensing was not accompanied by detectable cellular stress responses. However, other methods like detection of mitochondrial reactive oxygen species (mROS) stress responses (46) or inhibition of the p38 dephosphorylation by MAPK phosphatases (MKPs) might provide additional insights and therefore we cannot fully exclude minor sorterinduced cellular stress responses or an inhibition of the p38 dephosphorylation as cellular responses to sorting.…”

Plasma cells: You are what you eat

“…23 We also found that each plasma cell subset had mostly distinct immunoglobulin repertoires, were generated contemporaneously through the course of an NP-Ovalbumin (OVA) immunization, and showed similar expression of CD93, a marker of mature plasma cells, indicating that these subsets are formed independently of each other. 155 These cells recover after a rest period ex vivo. These data suggest an intrinsic ability of long-lived plasma cells to robustly import nutrients, rather than changes in the nutrient content in the local microenvironments of long-and short-lived plasma cells.…”

“…One technical caveat we have noticed anecdotally is that plasma cells assayed immediately after having been fluorescence activated cell sorted display very poor glucose import rates, perhaps due to stresses imposed by the process. 155 These cells recover after a rest period ex vivo. Given the minimal expression of known glucose transporters in mouse and human plasma cells, as-yet unknown genes and pathways likely promote extracellular sugar import.…”

Rapid light-dependent degradation of fluorescent dyes in formulated serum-free media

“…Two recent studies showed that cell sorting can induce oxidative stress, altering the basal metabolic state of cells (17), and that the presence of photo-sensitive molecules within standard serum supplemented cell culture medium could drive morphological changes or cell death via oxidative stress (18). In each case, addition of serum or antioxidants reduced measurable cell stress.…”