Cell Viability Assay with Lactoferrin

Growth curves were performed as previously described^{23 (link)}. Briefly, the indicated cell lines were grown exponentially in 8-mL tubes with different μM concentrations of bLF or bLFcin (described in the corresponding figure legends) and cellular proliferation/viability measured at 6-, 12-, 24- and 48-h after treatment. The number of viable cells was determined by counting with a haemocytometer after addition of 0.4% Trypan blue, according to the instructions from the manufacturer. Results were analysed for statistical significance by two-way ANOVA with GRAPHPAD Prism 5 Data Analysis Software (GRAPHPAD Software Inc., La Jolla, CA, USA). Mean and standard mean values were calculated from at least three biologically and technically independent experiments. p < 0.001 was considered significant and was indicated by asterisks in the figures.