Myoglobin from equine skeletal muscle

A proliferation assay was performed using CyQuant Reagent (ThermoFisher), following the supplier’s instructions. Briefly, BSCs were seeded in a 96-well plate at a density of 500 cells/well. Cell culture media consisted of either standard proliferation media or proliferation media with added hemoglobin from bovine blood (Sigma, St Louis, MO, USA) or myoglobin from equine skeletal muscle (Sigma) in concentrations of 1, 3, or 5 mg/mL. Both proteins were provided by the supplier in the oxidized met redox form (metmyoglobin or methemoglobin). Four plates were prepared with 6 replicates per group (n = 6) and single plates were recovered after 1, 3, 5, and 7 days of incubation by aspirating the media and storage at −80 °C. 100 µL media was aspirated and replaced by fresh media after 4 days. When all plates were recovered, CyQuant working solution was prepared by diluting the supplied lysis reagent 1:20 in sterile H₂O, followed by the addition of the dye reagent to a dilution of 1:400. Plates were thawed at room temperature, and 200 µL of CyQuant working solution was added to each well. Fluorescence was measured at an excitation of 480 nm and emission of 520 nm with a spectrophotometer (Synergy H1, Biotek, Winooski, VT, USA). Cell number was calculated with a standard curve of cells seeded at a known density.