Nnc 55 0396

All the chemicals used in this study were purchased from Sigma-Aldrich Química S.A. (Madrid, Spain) unless otherwise indicated. Tyrode’s albumin lactate pyruvate medium (TALP; [18 (link)]) was supplemented with 1.10 mmol/L sodium pyruvate and 3 mg/mL bovine serum albumin (BSA; A9647). Dulbecco’s PBS (DPBS) was supplemented with 1 mg/mL PVA and 0.005 mg/mL red phenol. North Carolina State University 37 medium (NCSU37; [19 ]) was supplemented with 0.57 mmol/L cysteine, 1 mmol/L dibutyryl cAMP, 5 µg/mL insulin, 50 μmol/L β-mercaptoethanol, 1 mmol/L glutamine, 10 IU/mL equine chorionic gonadotropin (eCG; Foligon; Intervet International BV, Boxmeer, Holland), 10 IU/mL human chorionic gonadotropin (hCG; Veterin Corion; Divasa Farmavic, Barcelona, Spain), and 10% (v/v) porcine FF [20 (link)]. A fixation solution of glutaraldehyde was prepared at 0.5% in phosphate-buffered saline (PBS). A staining solution of bisbenzimide (Hoechst 33342; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, H1399) was prepared at 1% (w/v) in PBS. The P4 (P8783) solution was prepared in DMSO (D2650) at 1mg/mL and frozen at −20 °C until use. The solution of the CatSper channel inhibitor NNC 55-0396 (NNC; Cayman 17216) was prepared in DMSO at 8.8 mM and frozen at −20 °C until use.

Human Aβ_1-42 was purchased from Peptide Institute Inc. (Osaka, Japan), curcumin (Figure 1) from Thermo Fisher Scientific K.K. (Waltham, MA, USA), and GT863 (purity: 99.1%) (Figure 1) from Green Tech Co., Ltd. (Kyoto, Japan). Nicardipine was obtained from Merck KGaA, NNC 55-0396 from Cayman Chemical Company (Ann Arbor, MI, USA), and PD173212 from Abcam PLC. (Cambridge, UK). curcumin and GT863 were dissolved in DMSO, with a final concentration of 0.1%. The other chemical reagents used were commercially available special-grade products.

CBDA was purchased from THC Pharm GmbH (Frankfurt, Germany), and CBGV was purchased from Toronto Research Chemicals Inc. (Ontario, CAN). CBDVA, CBGVA, CBCA, and CBCVA were synthesised by the laboratory of Professor Michael Kassiou at the University of Sydney. CBCV was synthesised in-house. NNC 55-0396 and ML-186 were purchased from Cayman Chemical (Michigan, United States) and Enamine (New Jersey, United States) respectively. For patch clamp electrophysiology, CBGVA and CBDVA were purchased from Cerilliant Corporation (Texas, United States). Unless otherwise stated, all drugs were prepared as stock solutions in DMSO and diluted in HEPES-buffered low potassium Hanks Balanced Salt Solution (HBSS) with 0.01% bovine serum albumin (BSA, Sigma, Castle Hill, Australia). HBSS comprises (mM) NaCl 145, HEPES 22, Na₂HPO₄ 0.338, NaHCO₃ 4.17, KH₂PO₄ 0.441, MgSO₄ 0.407, MgCl₂ 0.493, Glucose 5.56, CaCl₂ 1.26; (pH7.4, osmolarity 315 ± 15 mOsmol). Final DMSO concentrations ranged between 0.1%–0.2%.