4.7. Human Red Blood Cell Haemolytic Assay

Haemolytic activity of detergents on human erythrocytes was determined as previously described [73]. Briefly, after washing three times with phosphate-buffered saline (PBS 5 mM phosphate buffer containing 150 mM NaCl, pH 7.4), red blood cells were isolated from heparinised human blood by centrifugation. Erythrocytes were diluted in PBS saline to reach an absorbance of 1.0 at 655 nm. In a sterile 96-well microtiter plate, already containing 100 μL of the diluted compound, an aliquot of 100 μL of erythrocytes was added to reach a final volume of 200 μL. Complete haemolysis (100% haemolysis) was ascertained by preparing wells with PBS saline containing 0.2% Triton X-100 and red blood cells. The absence of haemolysis (0% haemolysis) was verified by preparing wells with PBS saline and erythrocytes. The microtitration plate was incubated at 37 °C with gentle mixing for 20 min. The haemolysis was quantified spectrophotometrically at 655 nm by a microplate reader (iMark, BioRad, Milan, Italy). The haemolysis percentage was obtained using the following equation:

All experiments were done in triplicate.