Cm h2cfda

To assess mROS, 2 × 10⁵ neutrophils in black flat bottom 96-well plates in HBSS containing 25 ng/mL GM-CSF were pre-incubated for 1 h with 5 μM MitoSOX Red (Life Technologies, Carlsbad, CA, USA) at 37 °C. Then, cells were stimulated with ionomycin, alum, MPLA and different vaccines at indicated concentrations. mROS-induced fluorescence was measured as described [18 (link)]. To detect the production of cytosolic ROS (cROS), chloromethyl-2’7’-dichlorodihydrofluorescein diacetate (CM-H₂CFDA) (Molecular probes, Life Technologies, Carlsbad, CA) was used; 1 × 10⁶ neutrophils were incubated for 30 min in 1 mL of 1 μM CM-H₂CFDA in HBSS supplemented with 25 ng/mL GM-CSF at 37 °C and 5% CO₂ in Falcon tubes. After washing with PBS and 30 min incubation with the indicated stimuli in HBSS in 24-well plates, the cells were harvested and fluorescence was measured immediately by flow cytometry on a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (BD Biosciences).

Chloromethyl‐2′7′‐dichlorodihydrofluorescein diacetate (CM‐H₂CFDA) (Molecular probes, Life Technologies, Carlsbad, CA, USA) was used to detect the production of cytosolic reactive oxygen species (cROS). A total of 1 × 10⁶ neutrophils in 1 mL were incubated in 24‐well plates for 30 minutes with CM‐H2CFDA in RPMI plus 2% autologous plasma with 25 ng/mL GM‐CSF at 37°C and 5% CO_2. Cells were harvested, washed with PBS, and stimulated for 30 minutes with the indicated stimuli in HBSS at 37°C and 5% CO₂. Immediately thereafter, fluorescence was assessed by flow cytometry on a BD FACS Canto II and analyzed with FlowJo software.
MitoSOX Red (Life Technologies) was used to detect the mitochondrial superoxide production in a plate reader assay. A total of 2 × 10⁵ neutrophils in 200 µL were seeded into black flat bottom 96‐well plates (Thermo Fisher) in HBSS medium containing 25 ng/mL GM‐CSF and preincubated for 1 hour with 5 µM MitoSOX Red at 37°C and 5% CO₂ and then, different stimuli added. Induction of fluorescence by mROS was detected at 2 minutes intervals with a TECAN Infinite M1000 plate reader. The area under the curve was calculated and compared to the medium control.

ROS were determined by incubating 2 × 10⁵ K562^shRNA, K562^shFtH and K562^shFtH cells NAC-treated with 1μM redox-sensitive probe 2′-7′-DCF (CM-H2CFDA; MolecularProbes, Eugene, OR, USA) for 30 min at 37°C. Afterward, pellet was washed twice with 1X PBS, then the pellet was resuspended in 1X PBS and analyzed using a FACS BD LSRFortessa^TM X-20 cytofluorometer (BD Biosciences).

ROS were determined by incubating H460^siRNA and H460^siFHC with the redox-sensitive probe 2’-7’-DCF (CM-H₂CFDA; Molecular Probes). In detail, 1 × 10⁶ H460 cells were plated in 96-well plates and incubated with Hanks balanced saline solution (HBSS), 10 mm glucose and 20μm DCF for 15 min at 37°C. After two cyclewashes, cells were maintained in HBSS supplemented with 10 mm glucose. Fluorescence was revealed, after 60 min incubation, using the Victor3 Multilabel Counter (Wallac, Perkin Elmer) at 485 nm and 535 nm for excitation and emission, respectively. Results were normalized on protein concentration evaluated by the bicinchoninic acid (BCA) method (Thermo Fisher Scientific).

ROS were determined by incubating MCF-7 and H460 silenced and unsilenced cells with the redox-sensitive probe 2′-7′-DCF (CM-H2CFDA; Molecular Probes, Eugene, OR) [32 (link)]. Briefly, 1 × 10⁶ cells were plated in 96-well plates and incubated with Hanks balanced saline solution (HBSS), 10 mM glucose and 20 μm DCF for 15 min at 37 °C. After two cycle washes, cells were maintained in HBSS supplemented with 10 mM glucose. Fluorescence was measured using the FACS FORTESSA.