CellROX™ Deep Red Reagent, for oxidative stress detection

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 10^6 cells were seeded.	- Cells were treated with 5 µM CellROX Deep Red or 10 µM MitoPY1 in HBSS containing 2.5 mM of probenecid.

Upstream tips
- 10^6 cells were seeded.
Protocol tips
- Cells were treated with 5 µM CellROX Deep Red or 10 µM MitoPY1 in HBSS containing 2.5 mM of probenecid.

Publication protocol

Flow cytometry was performed according to previous reports following generation of a single-cell suspension (24). Unless otherwise noted, all antibody and streptavidin staining was carried out on 106 cells for 15 minutes at 4 C in in PBS with 2% fetal bovine serum (FBS). To measure total or mitochondrial ROS, cells were stained for surface markers, then loaded with 5 µM CellROX Deep Red (Molecular Probes) or 10 µM MitoPY1 (Tocris) in HBSS containing 2.5 mM of probenecid (Molecular Probes) followed by incubation for 30 minutes at 37°C. Following incubation, cells were washed twice, kept ice-cold in HBSS/probenecid, and analyzed immediately. To measure superoxide production, cells were incubated with 2.5 µM DHE (Molecular Probes) in HBSS for 30 minutes at 37°C followed by two washes and immediate analysis similar to CellROX and MitoPY1. For measurement of mitochondrial potential (ΔψMt), antibody-stained cells were placed in 40 nM TMRM suspended in phenol red-free DMEM supplemented with 5% FBS, L-glutamine, sodium pyruvate and non-essential amino acids. Cells were incubated with TMRM for 30 minutes at 37°C. Staining was quenched by placing samples on ice and ΔψMt was immediately assessed. To measure total and respiring mitochondrial mass, cells were stained for cell surface markers, then incubated with 50 nM MitoTracker Green and/or 5 nM MitoTracker DeepRed (both from Molecular Probes) in PBS with 10% FBS for 30 minutes at 37°C. After incubation, cells were washed twice and kept on ice in PBS with 10% FBS until analysis. Apoptosis was measured using AnnexinV-APC in 1× AnnexinV staining buffer according to manufacturer’s instructions (BD Biosciences). For intracellular IFN-γ and TNF-α staining, splenocytes were incubated for 6 hours on 96-well plates coated with 2.5 ug/ml CD3/CD28 antibodies (eBioscience), in the presence of 1 µM Brefeldin A. Cells were then stained for membrane antigens, fixed and permeabilized using FoxP3 fixation/permeabilization kit (eBioscience), and stained with anti-cytokine antibodies for 40 minutes at 4°C. For granzyme B (GzmB) and GLUT1 detection, spleen cells were stained for surface antigens, fixed/permeabilized and stained with PE-conjugated anti-GzmB antibodies or unlabeled rabbit anti-mouse GLUT1 antibody followed by APC-conjugated goat anti-rabbit IgG Fab fragment. Flow cytometry data was acquired using FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software, version 7.6.1 (Tree Star).

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