Thioltracker violet

Fibroblasts, treated or not with 1 mM TCEP (tris[2-carboxyethyl]phosphine) for 30 min at 37 °C, were incubated with 20 μM ThiolTracker Violet (Invitrogen) for 30 min at 37 °C, washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. The images were acquired by Zeiss Axiovert 135 TV fluorescence microscope, and the ThiolTracker Violet signal was quantified by ImageJ software. iNs were incubated with 20 μM ThiolTracker Violet (Invitrogen) for 30 min at 37 °C, washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. The cells were then permeabilised for 3 min in PBS containing 0.1% Triton X100 and 10% normal goat serum. Next, the cells were incubated with Alexa Fluor 647 mouse anti-human CD56 (N-CAM, BD Biosciences, diluted 1:40) for 1 h at 37 °C, and with 2 μg/ml Hoechst for 2 min. After washing, the cells were analysed by IN Cell Analyzer 1000 system (GE Healthcare). The ThiolTracker Violet fluorescence in N-CAM-positive cells was collected to compare relative glutathione contents.

Glutathione content was measured under normoxic conditions and after exposure to 30 min and 6 h hypoxia using the GSH-Glo Assay Kit (Promega, Madison, WI, USA) according to manufacturer instructions. Luminescence was measured using a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Changes in GSH levels were also measured in hypoxic live cells using ThiolTracker Violet (Invitrogen, catalog # T10096). Cells were loaded with 20 µM ThiolTracker Violet glutathione detection reagent and 100 nM SYTO 16 Green Fluorescent Nucleic Acid Stain (Invitrogen, catalog # S7578) and imaged within the hypoxic environment using an LS620 microscope (Etaluma, Carlsbad, CA, USA) fitted with a 20 × objective at 0, 30 min, and 6 h at 1% O₂. Fluorescence intensity was quantified in 12 random cells per field using FIJI v2.1.0.
Intracellular succinate concentration was measured in cells under normoxic conditions and after exposure to 1- and 6-h hypoxia using a commercial assay (Sigma catalog # MAK184, St. Louis, MO, USA) according to manufacturer instructions. Absorbance at 450 nm was measured using a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Succinate concentrations per well were normalized to protein content using a Pierce Rapid Gold Protein Assay (Pierce, Rockford, IL, USA).

Cells were washed with PBS (–) and treated with 5 μM CellROX Oxidative Stress Reagents (cat. no. C10444; Life Technologies, Carlsbad, CA, USA) for ROS staining, or 20 µM ThiolTracker Violet (cat. no. T10095, Molecular Probes, Eugene, OR, USA) for GSH staining.
Fluorescence images were acquired using an EVOS™ M5000 imaging system (Thermo Fisher Scientific Inc.). Fluorescence intensity was quantified using ImageJ software v1.52A (National Institutes of Health; http://imagej.nih.gov/ij/). For corrected total cell fluorescence (CTCF), we used the following formulas^{61 (link)}:

CTCF = integrated density (total area of selected cells × mean fluorescence of background readings)

CTCF per cell = CTCF/ Ncells

where “Integrated Density” is the integrated intensity of the pixels for all cells in the image, total cell area is the number of pixels of all of the cells, background fluorescence is the average mean gray value of nearby regions containing no cells, and Ncells is the number of cells that was measured by counting fluorescently labeled nuclei from images.