Diphenylene iodium dpi

Losartan, lisinopril and AngII were purchased from Wako Pure Chemical (Tokyo, Japan). Benazepril was purchase from Tokyo Chemical Industry (Tokyo, Japan). PD123319, ZD7155, A779, SR202 and EHT1864 were purchased from Tocris Bioscience (Bristol, UK). Diphenylene iodium (DPI) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) were purchased from Sigma-Aldrich. A water-soluble derivative of forskolin, 7-deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl]-forskolin (forskolin-ws), was purchased from Calbiochem (San Diego, CA, USA).
Antagonists of AT1R (losartan and ZD7155) and AT2R (PD123319), the Mas receptor (A779) and PPARγ (SR202) and inhibitors of ACE (benazepril and lisinopril), NOX (DPI and AEBSF) and Rac (EHT1864) were dissolved in the perfused solution and administered to the striatum through the probe during the experimental period. Forskolin-ws, which stimulates cAMP production by activating adenylyl cyclase^{48 (link)}, and AngII were dissolved in sterilized physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) and directly administered into the striatum through the thin needle of the MI-A-I-8-03 probe using an ESP-32 pump. The flow rate was 0.1 μL/min, and the total volume was 1 μL for forskolin-ws and 2 μL for AngII.

Neutrophils were seeded on glass cover slips coated with 0.001% poly‐L‐lysine (Sigma‐Aldrich) with a concentration of 1 × 10⁵ cells per well if not stated otherwise. PMNs were stimulated with 4 μM ionomycin (free acid, Sigma‐Aldrich), 100 nM phorbol 12‐myristate 13‐acetate (PMA, Sigma‐Aldrich), 15 μM Ece1 peptides including candidalysin (if not otherwise stated) or infected with C. albicans yeast (MOI 2) for a defined time period, following fixed using 2% paraformaldehyde (PFA) and stored at 4°C. In the infection experiments, the fungus was added to 1 × 10⁴ PMNs 1 h after the cells were seeded.
For the pathway studies, neutrophils were incubated for 30 min before stimulation with 10 μM BB‐Cl‐amidine (PADi, Cayman Chemicals), 15 μM diphenyleneiodium (DPI, Sigma‐Aldrich), 15 μM 4‐hydroxy‐TEMPO (TEMPOL, Sigma‐Aldrich), 10/20 μM BAPTA‐AM (Abcam), 15 μM SYK inhibitors R406 (InvivoGen) and 12.5 μM piceatannol (InvivoGen), 15 μM PI3K blocker wortmannin (InvivoGen), 2.5 μM AKT inhibitor XI (InvivoGen) or NLRP3 blockage using compound 1 μM MCC950 (InvivoGen).