2.3. Sulforhodamine B (SRB) cell viability assay

For SRB assay, 5–8×10³ cells (~70–80% confluency depending on the cell line) were seeded in 96-well plates in regular culture medium in triplicate, overnight. The following day, serial dilutions of complexes, Cu(Sal-Gly)(pheamine), Cu(Sal-Gly)(phepoxy) and Cu(Sal-Gly)(phen) (0.19–12.5 µM), were freshly prepared and added to the cells. After 24 or 72 h, viable cells were fixed with the 50% trichloroacetic acid (TCA) at a final concentration of 10%. Plates were kept at 4 °C for 1 h, the supernatant was discarded and the plate was washed with deionized water five times. TCA-fixed cells were stained with SRB solution (0.4% in 1% acetic acid) for 30 min at room temperature (RT). Unbound SRB was removed by washing with 1% acetic acid and air-dried. Bound SRB stain was solubilized with Tris base solution (10 mM, pH:10.0), and plates were left on a shaker (10 min, 150 rpm). Absorbance was read by a spectrophotometer at 570 nm.