Barbital

Classical complement pathway activation was assessed using E^shA in Gelatin Veronal Buffer supplemented with Ca²+ and Mg²+ (GVB++; 10 mM Barbital, 145 mM NaCl, 0.1% Gelatin, 0.5 mM MgCl2, 0.15 mM CaCl2, pH 7.3 ± 0.15; Boston BioProducts, Ashland, MA, USA) (Morgan, 2000 ). In brief, approximately 5 × 10⁶ E^shA were incubated with serially diluted NHS (20% to 2.5%) and different titrations (0–50 μM) of cisplatin or pyridostatin in GVB++ at 37ºC for 10 min. For negative controls, 5 mM EDTA was added to the buffer to inhibit complement activation. For the alternative pathway, rabbit erythrocytes (E^rabb) and Gelatin Veronal Buffer supplemented with Mg²+ & EGTA (GVB-Mg-EGTA; 10 mM Barbital, 145 mM NaCl, 0.1% Gelatin, 0.5 mM MgCl₂, 10 mM EGTA, pH 7.4 ± 0.2; Boston BioProducts,) were used (Morgan, 2000 ). All the other conditions were the same as that in the classical pathway. After incubation, the erythocytes were centrifuged after the incubation, and the optical density (OD) at 414 nm (OD414) was measured in each 80 μl aliquots of supernatants containing released hemoglobin and used to calculate the percentage of hemolysis as follows: Hemolysis rate (%) = [(A - B)/(C - B)] × 100%, where A is the OD414 of tested samples, B is the OD414 of negative controls with EDTA, and C is the OD414 of maximum hemolysis induced by H2O.