Cancer Cell Viability Assay Using SRB

The viability of cancer cells after treatment with the juice was determined using the Sulforhodamine B (SRB) assay, as previously described with few modifications [14 (link)]. SRB is a dye that binds to basic amino acids of cellular proteins and, then, the number of viable cells is estimated with colorimetric evaluation [15 (link)]. Cells were plated in 96-well plates and treated with different concentrations of the juice (0.0007–1% v/v). Then, the cells were fixed with the addition of 25 μL of 50% (w/v) cold trichloroacetic acid (TCA) (Applichem, Darmstadt, Germany) to the growth medium and incubation of the plates at 4 °C for 1 h. The cells were washed five times with tap water and then stained with 50 μL of 0.4% (w/v) SRB dye (Sigma-Aldrich, St Louis, MO, USA) in 1% (v/v) acetic acid (Scharlau, Barcelona, Spain) for 30 min at room temperature. Next, the cells were rinsed five times with 1% (v/v) acetic acid to remove the unbound dye. The fixed, stained plates were allowed to air-dry and afterwards the bound dye was solubilized by adding 100 μL of 10 mM Trizma base (Sigma-Aldrich, St Louis, MO, USA) for at least 5 minutes. Absorbance was measured at 570 nm using an ELISA plate reader (Sunrise, Tecan, Männedorf, Switzerland) and the percent cellular survival was calculated using the formula: