2.6.1. Sulforhodamine B (SRB) Assay

H9c2 cell mass was measured using sulforhodamine B (SRB) assay [45]. This test is based on the ability of SRB to bind to protein amino acid residues of fixed cells. The SRB sulfonic groups bind stoichiometrically to the essential amino acid residues in a slightly acidified medium, with the amount of SRB bonded to the cellular protein being directly proportional to the cell mass [46]. In order to assess the cytotoxic effect of the plant extracts in H9c2 cells and/or the effect on cellular proliferation, H9c2 cells were seeded in 48-well plates at the density of 35,000 cells per mL [1], and twenty-four hours after seeding, cells were treated with the plant extracts at the concentrations of 1 or 25 µg/mL for 6, 24, or 48 hours. After treatment, the culture medium was removed, and the cells were rinsed with phosphate-buffered saline (PBS). Then, cells were then fixed in ice-cold methanol, supplemented with 1% acetic acid, and stored at −20°C for 24 hours. Following this process, the methanol/acetic acid was removed, and the plates were dried at 37°C. Later, 250 µL of 0.05% of SRB in 1% acetic acid was added. The fixed cells were incubated with the SRB solution for 1 hour at 37°C. The SRB solution was then discarded, and the plates were rinsed with 1% acetic acid solution, to remove the excess of unbound SRB, dried at room temperature. A volume of 500 µL of 10 mM Tris buffer (pH10) was added to dissolve the protein-bound SRB. The optical density was measured at 540 nm using a Biotek Cytation 3 reader (Biotek Instruments, Winooski, VT, USA). In order to assess the cytoprotection of the plant extracts against Dox-induced H9c2 cell death, H9c2 cells were pretreated with the plant extracts at the concentrations of 1 or 25 µg/mL for 3 hours before Dox treatment. H9c2 cells were then treated with 0.5 or 1 µM of Dox for 24 hours, as previously described [1, 47]. After Dox treatment, the culture medium was removed and H9c2 cells mass was measured as described above.