Canagliflozin’s antihemolytic activity was measured as described previously [15 (link)]. S. suis SC19 from overnight culture was centrifuged to obtain the supernatant. The supernatant was filtered through a 0.22 filter (Millipore, Billerica, MA, USA). Various amounts of supernatant were added to defibrillated sheep erythrocytes. The minimum concentration that caused complete lysis of the red cells was selected. Using the same method, the hemolytic activity of the purified SLY protein was determined, and a concentration of 0.912 μg/mL was set as the dose for subsequent experiments. Canagliflozin solutions of different concentrations (0, 2, 4, 8, 16, and 32 μg/mL) were mixed with supernatant or 0.912 μg/mL SLY at 37 °C for 30 min. Then, a solution of 2% sheep red blood cells was added and incubated with the previous mixture at 37 °C for 30 min. Then we centrifuged the incubated cultures and collected the supernatant. The amount of heme in the supernatant was quantified by measuring the absorbance at 543 nm using a microplate reader. There was 100% heme release in red blood cells treated with 2.5% Triton X-100. The percentage of heme released in the sample is used to evaluate the anti-hemolysis effect of the drug.
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