A redox-based characterization of human immune cell subsets by polychromatic flow cytometry

Abstract

Cellular redox state determinants are traditionally studied using fluorescent microscopy and immunoblot analysis; however, no procedure has been developed for simultaneous measurement in various immune cell subsets. Here, we present a flow cytometry assay for measuring antioxidant defense systems and reactive oxygen species simultaneously in T, B, and natural killer lymphocytes. We describe steps for preparing and treating peripheral blood mononuclear cells, surface and dye staining, cell fixation/permeabilization, and intracellular staining. We then detail machine standardization, acquisition, and analysis.

Keywords: Cell Biology; Flow Cytometry; Immunology.

Graphical abstract

Figure 1

Sample and staining control preparation After thawing, PBMCs should be treated either with Menadione and/or H₂O₂, while some cells should be kept untreated. Following the treatment, cells should be divided into 15 mL tubes where 1) Untreated cells that will be unstained 2) Untreated but fully stained 3) Untreated but stained only with antibodies and dyes that do not require gating controls 4) H₂O₂ Treated cells that will be used for single stains for compensation of redox dyes 5) H₂O₂ treated cells fully stained 6) menadione treated cells fully stained.

Figure 2

Gating Strategy for flow cytometry analysis of redox makers on human immune cell subsets (A) Gating strategy to detect T, B and NK cell subsets. Singlets are defined by gating FSC-A against FSC-H. Live cells are identified by gating on viability dye (LD) negative cells . Morphological parameters FSC-A and SSC-A then define the Lymphocyte gate. T cells are identified as CD3+ cells. On the CD3+ cells, CD4 and CD8 markers allow to identify the major T cell populations (CD4+ helper T cells and CD8+ Cytotoxic T cells). In both CD4+ and CD8+ compartment, T naïve-like (NL, CCR7+CD45RO-), T central memory (CM, CCR7+CD45RO+), T effector Memory (EM, CCR7- CD45RO+), T terminal effector memory (TE, CCR7-CD45RO-) are defined. On CD3- cells, B cell are defined as CD19+. On CD19+ cells, B Naïve are defined as CD21+,CD27- (Q1), and B cell Memory are defined as CD21+,CD27+ (Q2). On CD3- cells, total NK cells are defined as CD56+ cells. From the CD56+ population we can move further into defining CD56BRIGHTand CD56DIM. (B) Redox markers represented by histograms. Compensated redox related markers on the total gate of lymphocytes with the red peaks defining the fully stained (MVG - MitoView Green, MSR - MitoSOX Red, Nrf2 - nuclear factor erythroid 2–related factor 2, SOD1 - superoxide dismutase 1, TTV -ThiolTracker Violet) and the gray peak defines their FMO controls.

Figure 3

Positive controls (A) Histograms showing the ratios amongst MSR and MVG in T, B and NK cells. Results from PBMCs treated with H2O2 (orange) and Menadione (purple) and untreated (green) are presented. (B) Ratio of MSR and MVG MFI values of three different healthy donors in each condition on total lymphocytes. Menadione demonstrates a significant increase in the ratio of MSR/MVG when compared to the untreated (P = 0.0044). (C) Histograms showing TTV levels in total Lymphocytes. Results from PBMCs untreated or treated with different concentrations of menadione are presented (20 μM – light blue, 50 μM – purple, 100 μM – fuchsia).

Figure 4

Visualizing Nrf2 by proteasomal degradation inhibition PBMCs were treated for 24 h with 5 μM and 10 μM of inhibitor MG132 of proteasomal degradation. (A) Morphology of lymphocytes untreated and treated with MG132. (B) Histogram overlays of MFI of Nrf2 on total lymphocytes in treated and untreated samples. The gray peak represents total lymphocytes treated with MG132 but not stained for Nrf2_APC while the blue peak represent PBMCs that have been treated wither with 5 μM (left histogram) or 10 μM (right histogram). As controls, we also include untreated (DMSO) cells in red. MFI values for each peak are represented in the table below each histogram plot.