Dhr 123

The production of reactive oxygen species (ROS) was evaluated by flow cytometry using the fluorogenic substrate dihydrorhodamine 123 (DHR 123, Cayman Chemical Company, Ann Arbor, MI, USA) as an indicator of production of ROS. Briefly, 5 × 10⁵ neutrophils were incubated with 1 μM of DHR123 for 15 minutes and after infected with L. (V.) braziliensis promastigotes or PMA (10 ng/mL) plus ionomycin (500 ng/mL) or buffer was added. Control samples containing no stimulus were runs in parallel. After 15 minutes at 37°C, 5% CO₂, cells were washed in PBS and analysed by flow cytometry. The neutrophil population was selected based on forward and side scatter followed by DHR123 fluorescence to determine the frequency and mean of fluorescence intensity (MFI) of double positive cells CD15⁺DHR⁺. Separate controls verified of this population, corresponded to neutrophils according to CD15⁺ surface stain. The FACS strategy to assessment of respiratory burst is shown in Figure 2A.

The IL-1β levels in the cellular supernatants and peritoneal lavages were measured using an enzyme-linked immunosorbent assay (ELISA) kit (DY201 or DY401, R&D SYSTEMS). LDH secretion was analyzed using an assay kit (BCT-LDHP, BIOMAX, Seoul, Republic of Korea). For the ROS measurement, LPS-primed BMDMs (1.25 × 10⁵ cells/well in a 96-well-black plate) were treated with dihydrorhodamine 123 (DHR123, CAYMAN CHEMICAL, Ann Arbor, MI, USA) in the presence of rotenone (160 μM) and JC for 6 h. The caspase-1 activity was determined using mouse recombinant caspase-1 (one unit per reaction, BIOVISION, Milpitas, CA, USA) and a caspase-1 fluorometric assay kit (BIOVISION) in the presence of JC or the pan-caspase inhibitor (Z-VAD-FMK, 10 μg/mL; R&D SYSTEMS). The recombinant caspase-1 was co-incubated JC or Z-VAD-FMK in the reaction buffer containing YVAD-AFC for 1 h at 37 °C according to the manufacturer's protocol. The plates were analyzed using a multi-microplate spectrophotometer (Synergy H1 Hybrid Multi-Mode Reader, BIOTEK, Winooski, VT, USA).

ICM contained (mM): KCl (120), NaCl (10), KH₂PO₄ (1), HEPES (20), sodium succinate (2), EGTA (1) and KOH (pH 7.1). The free [Ca²⁺] was adjusted to the desired level by varying the ratio of Ca²⁺/HEDTA, calculated using maxchelator (C Patton, Stanford University, CA, USA). Chemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA), except for DHR 123 (Cayman Chemical, Ann Arbor, MI, USA), CDNB (Alfa Aesar, Ward Hill, MA, USA) and Rhod-2 AM, TMRE and MitoSOX (Life Technologies, Grand Island, NY, USA). Recombinant His-tagged Mcl-1 was purchased from Proteintech Group, Inc. (Chicago, IL, USA). Peptides based on the human VDAC1 sequence were synthesized by Biomatik (Wilmington, DE, USA): control (LVLGYEGWLA), N-terminal (GLGKSARDVFTKGYGFG) and L14-15 (LAWTAGNSNTR). Cell-permeant versions were tagged with antennapedia-homeodomain-derived antennapedia (Antp; RQIKIWFQNRRMKWKK) at the carboxyl-terminal of each peptide.

L929sAhFas cells and BMDMs were seeded in 24-well suspension plates (0.1mln/ml and 0.4mln/ml accordingly) and stimulated for cell death. One hour before each time point, fluorescent probes were added to proper wells: 0.5 μM C11-BODIPY (ThermoFisher, MA, USA) or 1 μM DHR-123 (Cayman Chemical, MI, USA) and 0.5 μM of DRAQ7 cell death stain (BioStatus, Shepshed, UK). Lipid ROS and ROS accumulation was measured at specific time points using BDFortessa. Fluorescence was collected in B530 (C11-BODIPY and DHR123) and R780 (DRAQ7) channels. Only fluorescence of not permeabilized cells was analyzed. Gating strategy is presented in supplementary materials (Supplementary Fig 1).

Neutrophil respiratory burst was assessed based on oxidation of the fluorescent dihydrorhodamine 123 probe (DHR 123; Cayman) as previously described (Sarantis and Gray-Owen 2012 (link)). In brief, MPRO 2.1 cells were plated in 24-well dishes and grown until reaching 80% confluency; at that point, the medium was removed and the cells were then exposed to 50 μg AgNP/ml for 24 h. Later the cells were then washed with calcium (Ca²⁺)-free Hanks balanced salt solution (HBSS) and re-suspended in DHR 123 (10 μM)-containing HBSS (with Ca²⁺) for 15 min at 37 °C, protected from any light. The cells were then treated with 1 μg PMA/ml (or medium for basal activity measures) for 15 min at 37 °C, again protected from any light. Cells were then collected, centrifuged/washed, and then the total fluorescence was measured in the BD Accuri™ C6 flow cytometer. Cells were gated to remove any debris or dead cells; 10, 000 events were counted.

Blood was collected from healthy human volunteers (n = 3–6; Etablissement Français du Sang, EFS, Clermont-Ferrand, France). Donors gave their written informed consent for the use of blood samples for research purposes under EFS contract n°16-21-62 (in accordance with the following articles L1222-1, L1222-8, L1243-4 and R1243-61 of the French Public Health Code). Blood leukocytes were obtained by hemolytic shock using ammonium chloride solution. Leucocytes were then washed with RPMI 1640 medium (GIBCO, ThermoFisher Scientific), centrifuged and suspended in supplemented RPMI (fetal bovine serum (FBS) 10%, gentamicin 50 μg/mL and glutamine (Gln) 2 mM). Cells were placed in 96-well plates (Cell Wells™, Corning, NY, USA), incubated with the extracts or the compounds and dihydrorhodamine 123 (DHR 123, 1 μM, Cayman Chemical Company, Ann Arbor, MI), and stimulated, or not, by 1 µM phorbol myristate acetate (PMA) for 60 min. The fluorescence intensity of rhodamine 123, which is the product of dihydrorhodamine 123 oxidation by ROS, was recorded (excitation/emission: 485/538 nm) using a microplate fluorometric reader (Tecan Spark®, Mämmedorf, Switzerland) [33 (link),34 ].