Mitotracker orange

Metabolic activity was investigated by incubating the cells for 4 h with resazurin (final concentration 100 μM; Alfa Aesar) after two hours of incubation posttreatment. Metabolically active cells transform nonfluorescent resazurin into fluorescent resorufin, which can be quantified using a multiplate reader (F200; Tecan) at λ_ex 560 nm and λ_em 590 nm. Absolute sample values were normalized to that of untreated cells = 100%. To quantify mitochondrial mass, cells were incubated for 15 min with chloromethyltetramethylrosamine, also called MitoTracker Orange (MTO; final concentration 1 μM; Thermo Fisher), at 6 h after plasma treatment. The cationic rosamine probe only binds to mitochondrial membranes with intact potential. Sample acquisition was performed using flow cytometry. To quantify nonterminally dead (alive) and terminally dead cells, DAPI was used to discriminate the percentage of either population using flow cytometry. For some experiments, the amount of cells active for caspase 3 and 7 was investigated to quantify the amount of apoptotic cells. For this, cells were incubated for 30 min with CellEvent dye (final concentration 2.5 μM; Thermo Fisher). Samples were analyzed by flow cytometry.

Cells were grown on glass coverslips for 24 h in a complete medium. Then, cells were fixed with paraformaldehyde 4% in phosphate buffer saline solution (PBS), stained with 20 nM Mitotracker Orange (ex: 554 nm/em: 576 nm) (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. Images were acquired by confocal microscopy LSM 800 and software ZEN 2.1 60× objective and analyzed by ImageJ version 1.53t (U.S. National Institutes of Health, Bethesda, MA, USA).

We cultured COS7 cells expressing a stably integrated histone H2B-HaloTag plasmid²² (gift from Tim Brown at Janelia Research Campus, RRID:CVCL_0224) in Dulbecco’s Modified Eagle Medium (Gibco 11965-092) with 10% FBS (VWR: 1500-050) and 1% (v/v) 10,000 U/ml Penicillin-Streptomycin (Gibco 15140-122). We obtained iPSC cells (purchased from the Coriell Institute for Medical Research, RRID:CVCL_IR34) from the Allen Cell Collection Cell Lines (Mono-allelic mEGFP-tagged TUBA, AICS-0012) and cultured them in basal media with provided 5x supplement with a ratio of 4:1 (STEMCELL Technologies 85850_c) and 1% (v/v) 5000 U/ml Penicillin/Sreptomycin (Gibco 15070-063). For fixed cell imaging, we incubated COS7 with either 250 nM mitotracker orange (Thermo Fisher, M7510) in culture media for mitochondria staining or 250 nM JF549 in culture media for histone staining for 30 min. We then fixed cells with 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) and 8 nM/ml sucrose (Sigma, S7903) in cytoskeleton buffer (composed of 10 mM MES, 138 mM KCl, 3 mM MgCl and 2 mM EGTA) for 20 min at room temperature. For actin staining, we permeabilized cells in 0.2% Triton-X (VWR Life Science, 0694) for 10 min, then blocked with 2% bovine serum albumin (Sigma, A9418) and 0.1% Triton-X for 10 min before staining with phalloidin 555 (Thermo Fisher, A34055) for 20 min.

Immunofluorescence staining was performed by seeding cells at a density of 5 × 10⁴ cells/well on a 15 mm poly-L-lysine-coated cover glass in a 6-well plate, followed by a 24 h incubation at 37 °C. The cells underwent fixation with 4% paraformaldehyde, permeabilization with 0.1% Triton X-100 for 15 min at room temperature, and triple washing with phosphate-buffered saline (PBS). Subsequently, cells were blocked with 1% bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) for 60 min at room temperature. Following three 10 min washes with PBS, the samples were incubated overnight at 4 °C with an anti-BRD4 monoclonal antibody. After three 3 min PBS washes, cells were exposed to Alexa Fluor Plus 488 conjugated Goat anti-rabbit immunoglobulin G (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. Mitochondria were stained with MitoTracker Orange (1:5000, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature. Visualization and photography of fluorescence-labeled HOXB9 and mitochondria were conducted using a fluorescence microscope.

MDSCs were incubated with 200 nM MitoTracker Green (ThermoFisher Scientific, M7514) or 500 nM MitoTracker Orange (ThermoFisher Scientific, M7510) for 15 min. Then flow cytometry was performed by using a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed with software FlowJo V10.