Bsa a7906

Mouse anti-LAMP1 monoclonal antibody (555798) was purchased from BD Biosciences; mouse anti-diphosphorylated ERK1/2 monoclonal antibody (M8159) was from Sigma-Aldrich; rabbit anti-ERK1/2 polyclonal antibody (V114A) was from Promega; mouse anti-β-actin monoclonal antibody (G043) was from Abm; mouse anti-γ-tubulin antibody (T6557) was from Sigma-Aldrich; goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) were from Santa Cruz Biotechnology; goat anti-mouse IgG-TRITC antibody (T5393) was from Sigma-Aldrich; BSA (A7906) was from Sigma-Aldrich; SDS-PAGE reagents were from Bio-Rad; fetal bovine serum (FBS) was from Gibco; LysoTracker (L7528) was from Thermo Fisher Scientific; fibronectin (F2006) was from Sigma-Aldrich; FGF2 was from PeproTech; and Alcian blue dye (74240) was from EuroDiagnostica. The recombinant NK1 fragment of HGF was produced using yeast Pichia pastoris expression system and purified with heparin affinity chromatography as previously described.³⁹ (link)

Non-commercial HEPES-buffered HTF (4.67 mM KCl, 0.31 mM KH₂PO₂, 90.69 mM NaCl, 1.20 mM MgSO₄, 2.78 mM glucose, 1.6 mM CaCl₂, 0.51 mM sodium pyruvate, 60 mM sodium lactate, 23.8 mM HEPES, and 10 μg/ml Gentamicin, (pH 7.4 with NaOH) was used to measure human sperm membrane potential. Non-commercial HEPES-buffered HTF was supplemented the same day with 25 mM NaHCO₃ and 0.5% w/v BSA A-7906 (Sigma) for capacitating conditions, and only with 0.5% w/v BSA for non-capacitating conditions. IVF samples were processed in commercial Quinn’s Advantage Human (HTF) fertilization media [CooperSurgical (Trumbull, CT, United States)] supplemented with 3 mg/ml human serum albumin (CooperSurgical) and pre-incubated overnight at 37°C with 5% CO₂ to equilibrate pH. The same media was used to capacitate IVF and donor sperm samples.
Mouse capacitating modified (TYH) medium [119.30 mM NaCl, 4.70 mM KCl, 1.71 mM CaCl₂, 1.20 mM KH₂PO4, 1.20 mM MgSO₄, 0.51 mM sodium pyruvate, 5.56 mM glucose, 20 mM HEPES, 15 mM NaHCO₃ and 5 mg/ml BSA A-2153 (pH 7.4 with NaOH)] was used to capacitate mouse sperm.