O phenanthroline

Folin–Ciocalteu’s phenol reagent (cat# 47742); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) (cat# 10102946001), O-Phenanthroline (cat# 516705) and the Griess reagent (cat# 516705) were purchased from Sigma (St Louis, MO, USA). Griess reagent, ACROS Organics (cat# AC328670500); Sodium persulfate (cat# AC202025000) and 1, 10-Phenanthroline (cat# P69100) were purchased from Fisher Scientific (Pittsburgh, PA, USA). All purchased chemicals were used without further purifications.

ALN was gifted by the scientific office of Riyadh Pharma (Riyadh, Kingdom of Saudi Arabia). Its purity was evaluated as per the official USP method [30 ] and found to be 99.95%. Acetic acid, o-phosphoric acid, iron (II) ammonium sulfate, boric acid, sodium hydroxide, potassium chloride, ethanol, and tetrahydrofuran (THF) were purchased from Prolabo, France. O-phenanthroline, DOP, NPOE, and DBS were procured from Sigma, Germany. PVC (HMW grade) was purchased from Fluka Chemie, Germany. Carbon electrodes (C-E-02-ELEC) were purchased from American Elements (Orlando, FL, USA). Deionized water was acquired from Aquatron Automotive water still A 4000, Bibby Sterillin Ltd., (Staffordshire, UK). Ferroin (o-phenanthroline-iron (II) complex) was made by mixing 100 mg of o-phenanthroline and 20 mL of 1 × 10⁻² M iron (II) ammonium sulfate. A clear solution of ferroin was then obtained by adding water and ethanol dropwise. Britton Robinson buffer (BRB) was prepared by stirring equivalent amounts of acetic acid (0.04 M), boric acid (0.04 M), and o-phosphoric acid (0.04 M). A pH range of 4 to 12 of BRB was prepared by adding 0.2 M sodium hydroxide dropwise [31 ].

Calpain protease activity imaging was performed with CMAC, t-BOC-Leu-Met (Molecular Probes™) in live DRG neurons cultured at 3 DIV. Neurons were incubated in a sequential two-step process with 200 nM of Mitotracker deep red for 15 min at 37°C and 10 μM CMAC, t-BOC-Leu-Met for 15 min at 37°C. Fluorescence (emission 430 nm), corresponding to cleaved t-BOC produced by calpain activity, was monitored in live neuronal imaging using a 40× objective on a Leica TCS SP8 laser-scanning confocal microscope. Confocal imaging settings were kept constant between genotypes and experimental replicates. 2D reconstructions of the stacks were performed with ImageJ (NIH, Bethesda, MD, USA). Cultured neurons were identified by morphological criteria and fluorescence intensity relative to area was measured in neuronal somas with ImageJ. At least 46 neurons were analyzed in three or more independent experiments for each treatment and genotype. For experiments using [Ca²⁺]_cyt modulators, DRG cultures at 2 DIV were treated for 24 h with: (i) extracellular calcium chelator, 1 mM EGTA (Sigma-Aldrich); (ii) intracellular calcium chelator, 20 μM BAPTA-AM (Molecular Probes™); (iii) inhibitor of metal-ion-dependent enzymes, 100 μM o-phenanthroline (Sigma-Aldrich); and (iv) two-fold increase physiological extracellular calcium, 3.6 mM CaCl₂.

CCCP (C2759, Sigma), Puromycin (P8833, Sigma), MitoBloCK-6 (5.05759.0001, EMD Millipore), MitoBloCK-10 (HY-115467, MedChemExpress), FuGENE HD Transfection Reagent (E2311, Promega), Oligomycin A (75351, Sigma), Antimycin A (A8674, Sigma), MG132 (1748, Tocris), o-phenanthroline (516705, Sigma), AEBSF (A8456, Sigma), TMRE (T669, Thermo Fisher Scientific) and trans-Resveratrol (70675-50, Cederlane).

Methanol, n‐Hexane, petroleum ether, ethyl acetate, Bouin's fluid, formalin, DPPH, O‐phenanthroline, potassium ferricyanide, tri‐chloroacetic acid, EDTA, and sodium dihydrogen phosphate were procured from Sigma‐Aldrich (St. Louis, USA). Diazepam and Fluoxetine hydrochloride were purchased from Square Pharmaceutical Ltd. (Dhaka, Bangladesh). Tween‐80 was obtained from Scharlab (Sentmenat, Barcelona, Spain). Aluminum chloride, Folin–Ciocalteu reagent, potassium acetate, sodium carbonate, gallic acid, isopropyl alcohol, ferric chloride, ferrous sulfate, sodium salicylate, hydrogen peroxide, and quercetin were bought from Merck (Darmstadt, Germany).