Disbac2 3

Estimation of ΔΨ_p was monitored by measuring the increase in absorbance of bis-(1,3-diethylthiobarbituric acid) trimethine oxonol [DiSBAC₂(3)] (Invitrogen) as previously described [24] (link). Briefly, L. amazonensis promastigotes (2×10⁷/mL) were added into black polystyrene 96-well microplates in HBSS+Glc containing 0.2 µM DiSBAC₂(3) in a final volume of 100 µL per well. The plate was incubated at 25°C in a microplate reader (POLARstar Omega, BMG Labtech) and fluorescence was recorded (λ_ex = 544 nm; λ_em = 584 nm) every 2 min. After signal stabilization raloxifene was added to final concentrations of 15, 30 or 60 µM. Gramicidin D 8 µM (Sigma-Aldrich) was used as a positive control. Untreated parasites and parasites incubated with the highest volume of diluent (DMSO 0.6%) were used as negative controls. No interference in DiSBAC₂(3) fluorescence was observed when raloxifene was added to HBSS+Glc in the absence of cells. Three independent experiments were performed, each one with triplicate samples.

Plasma membrane potential was determined using the slow-response potential-sensitive dye DiSBAC2(3) (Invitrogen, Life Technologies, Carlsbad, CA). Lin(−) cells or BMDDCs were harvested, washed three times with PBS, and re-suspended at a concentration of 0.5×10⁶ cell/ml in PBS with 100 µM CaCl₂. Cell suspensions were incubated with 5 µM (final concentration) DiSBAC2(3) in a quartz cuvette under constant stirring at 37°C for 3 min in the dark. Then the kinetics of the emission at 560 nm (Ex = 530 nm) were monitored by a QM-4 spectrofluorometer. The emission of each sample was recorded for 100 seconds before and until 500 seconds after the addition of 100 ng/ml CXCL12. Representative traces of three independent experiments were averaged and smoothed using standard Sigma-Plot 10.0 software (Systat Software, San Jose, CA).

A voltage sensor probe (VSP) set (CC2-DMPE and DisBAC₂(3)), VABSC-1 (Voltage Assay Background Suppression Compound), and Pluronic^®-F127 were purchased from Invitrogen (Carlsbad, CA, USA). BTX and TTX were purchased from Latoxan (Valence, France). Asante NaTRIUM Green-2 (ANG-2) was purchased from Interchim (Montluçon, France). All other reagents (Ponceau 4R) and solvents were obtained from Sigma-Aldrich (Saint-Louis, MO, USA) or Fisher Scientific (Waltham, MA, USA).

Voltage sensor probes, coumarin-labeled phospholipid CC2-DMPE (FRET donor) and oxonol dye DiSBAC₂(3) (acceptor) were from Invitrogen. Cells (3×10⁴ cells/well) in optical 96-well plates were loaded with 10 µM CC2-DMPE in FRET buffer (10 mM Hepes-NaOH, pH 7.4, 160 mM NaCl, 0.9 mM CaCl₂, 1 mM MgCl₂, and 10 mM glucose) containing 200 µg/ml Pluronic F-127 for 30 min, washed and incubated in 100 µl 10 µM DiSBAC₂(3) in FRET buffer for 20 min. Tartrazine (1.2 mM final concentration) was added, and after 10 min, fluorescence measurements (λ_ex of 420 nm; λ_em of 460 nm and 550 nm; 10-nm bandpass filters) were conducted in well mode at 37°C and 5% CO₂. Gain was adjusted to yield similar baseline readings for each fluorophor at resting potential. Following baseline acquisition, 10 µl 0.82 M KCl, 1 mM BzATP or buffer control were injected while monitoring fluorescence. Following subtraction of signal without cells, the signal ratio (SR) before and at equilibrium after depolarization was calculated, and the response ratio (RR) derived as RR=SR^depol/SR^pol.

DMEM medium, Neurobasal A medium, F-12 medium, Fetal Bovine Serum (FBS), HBSS, D-PBS, Penicillin/Streptomycin, B-27, Collagenase II, Dispase II, Trypsin, Alexa Fluor^® 647 conjugated anti-rabbit antibody, and DiSBAC₂(3) were purchased from Life Technologies (Carlsbad, CA, USA). DNAse 1, 30% BSA, Cytosine β-d-arabinofuranoside hydrochloride (ARA-c), 5-Fluoro-2′-deoxyuridine 5′-monophosphate sodium salt (FdU) solution (FdU), poly-d-Lysine, DAPI, and 10% neutral buffered formalin solution were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Angiotensin II was purchased from Tocris (Bristol, UK). C21 (MW = 475.2) and EMA401 (MW = 507.2) were synthesized by AGV discovery (Clapiers, France), purified by HPLC (>95%), and characterized by 1H NMR (Figures S3 and S4) and mass spectrometry. Anti-rabbit β3-Tubulin (D71G9) XP^® (anti-Tuj1) was purchased from Cell Signaling Technologies^® (Danvers, MA, USA). Micro-titer plate (384-well), µClear^®, was purchased from Greiner Bio-One SAS (Les Ulis, France). Purified mycolactone was obtained and quantified as described in a previous study [9 (link)], and re-suspended in DMSO at 5 mg/mL and stored at −20 °C as aliquots in amber glass tubes.

ZnONPs in powder form (catalog #544906; particle size 50–70 nm) were purchased from Sigma-Aldrich (Oakville, ON, Canada). An aqueous dispersion of AgNPs (2 mg/mL; 31% silver content; particle size 1–10 nm) was purchased from Sciventions Inc (Toronto, ON, Canada). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), sodium azide, glucose, MES (2-morpholinoethanesulfonic acid), DMSO (dimethyl sulfoxide), geneticin (G418), CCCP (carbonyl cyanide 3-chlorophenylhydrazone), and MUG (4-methylumbelliferyl-D-galactopyranoside) were purchased from Sigma-Aldrich. DiSBAC₂(3) [bis-(1,3-diethylthiobarbituric acid)trimethine oxonol] was purchased from Life Technologies (Carlsbad, CA, USA).