Carboxy ptio

Different ROS and RNS scavengers were used to evaluate the effect of radical stress inhibition on aPI efficacy. The scavengers included: 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) chloride (FeTPPs) (1 and 0.1 mM, Calbiochem) as a ONOO⁻ scavenger, 4,5-dihydroxy-1,3-benzenedisulfonic acid disodiumsalt hydrate (Tiron) (1.0 and 10 mM, Sigma-Aldrich, St. Louis, MO, USA) as a O₂^•− scavenger, sodium pyruvate (0.1, 1.0 and 10 mM, Sigma-Aldrich) as a hydrogen peroxide scavenger, carboxy-PTIO (0.2 and 2.0 mM, Cayman chemical, Ann Arbor, MI, USA) as a NO^• scavenger, D-mannitol (100 mM, Sigma-Aldrich) as a hydroxyl radical scavenger and sodium azide (1.0, 10 mM and 1.0 M, Sigma-Aldrich) as an ¹O₂ scavenger. Scavengers were added to fungal suspensions immediately before initiation of aPI and incubated for 1 h with RPMI 1640 without phenol red plus 2% glucose at 28°C. To evaluate fungal viability, 150 μL of the fungal suspensions were plated onto PDA, and incubated at 28°C for 72 hours [12 (link)].

Imaging of NO and CBF was as described [28 (link)]. For NO, ALI cultures were loaded with DAF-FM by incubation in 10 μM DAF-FM diacetate (ThermoFisher) on the apical side in HBSS plus 5 μM carboxy-PTIO (to scavenge baseline NO; Cayman Chemical) for 90 min. For submerged CFBEs on 8-well chambered coverglass (CellVis, Sunnyvale, CA, USA), loading was the same but for 45 min instead of 90 min. After washing, imaging was performed using an IX-83 microscope (10x 0.4 NA PlanApo objective, Olympus Life Sciences, Tokyo, Japan) with LED illumination (Excelitas Technologies LED120Boost), 16-bit Orca Flash 4.0 sCMOS camera (Hamamatsu, Japan), standard FITC filter set (470/40 nm excitation, 495 lp dichroic, and 525/40 nm emission; 49002-ET, Chroma Technologies) and MetaFluor (Molecular Devices, Sunnyvale, CA, USA). For cilia beating, cultures were maintained at ~28 °C in DPBS (+1.8 mM calcium) on the apical side and HEPES-buffered HBSS supplemented with 1× MEM amino acids (Gibco) on the basolateral side. Sisson-Ammons Video Analysis software was used to measure whole-field CBF. Diphenhydramine and all other reagents used were from Millipore Sigma unless otherwise specified.