2.4. Hemolysis Assay

A group of volunteers (n = 8) was formed to determine hemolysis by CB[6,7,8]. The separation of erythrocytes from plasma was performed by 2.7 rpm centrifugation with ficoll (it provides the density gradient) for 25 min. After plasma removal, the erythrocytes were washed two times with PBS by centrifugation 800× g for 10 min. Then, the erythrocytes were suspended in PBS to obtain 2% hematocrit. The erythrocytes were used immediately.

Next, a 2% RBC suspension was treated with CB[6] and CB[7] (CB[n] diluted in PBS) at concentrations of 0.2, 0.5, 1 and 2 mM and CB[8] at 0.01 in PBS, albumin (40 g/L), or an autologous serum from donors. Every sample was incubated 1 h by a shaker in a thermostat at 37 °C. The positive control was treated with Triton-100 (10 v/v) and the negative control was included in RBC in different biologically relevant media (PBS, albumin, or serum). After incubation the samples were centrifuged to 3.5 rpm × 15 min. The supernatant was transferred to wells to measure the absorbance of release hemoglobin at 540 nm. The level of hemolysis was calculated using the formula:

Abs—absorption index for the test sample;

Ab₀—absorbance index for negative control (corresponding buffer);

Abs₁₀₀—Absorption index for positive control (Triton X-100).