2.7.2. Hemolysis experiment

Irritating chemicals can cause erythrocyte membrane damage and lead to hemoglobin leakage; therefore, the content of oxygenated hemoglobin was quantified by hemolysis to evaluate the damage effects on the eye tissue of the subjects. Blood samples (2 mL) from the ear veins of healthy rabbits were gently stirred, and 2 mL of PBS was added. After centrifugation for 5 min, the supernatant was discarded. Ten milliliters of PBS were continuously added, and after 5 min of centrifugation, the supernatant was discarded. The above operation was repeated 4–5 times until the supernatant was without an obvious red color. The resulting red blood cells were diluted with PBS to form a red blood cell suspension with a volume fraction of 2%. PBS (negative control group), deionized water (positive control group), and Betoptic and MIDFDS (sample groups to be tested) were added to 0.2 mL of the erythrocyte solution, incubated at 37 °C for 4 h and centrifuged for 5 min. The supernatant color was observed. From each group, 100 µL of the supernatant was placed in a 96-well plate, the absorbance was measured with an enzyme marker (570 nm), and the hemolysis rate was calculated (Ablamowicz & Nichols, 2016).