0.22 filter

To screw vials holding 3 mL of different media, an excess of GCZ and freeze-dried NS were added individually (triple-distilled water, acetate buffer, phosphate buffer, and 0.1 N hydrochloric acid) and sonicated for 2 to 3 min to disperse the drug. The vials were maintained at 37 °C for 48 h on an incubator shaker (SI 300 UK) and centrifuged at 7500 rpm for 10 min. A Millipore 0.22 filter was used to separate and purify the clear liquid, and the GCZ concentration was evaluated using a UV–visible spectrophotometer set to 226 nm [48 (link),49 (link)].

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.

U87MG glioblastoma cells culture, lentivirus preparation and titration were as previously described.30 (link) U87MG cells were transduced with lentivirus particles in a medium supplemented with polybrene (16 µg/mL; hexadimethrine bromide, Sigma). Media were changed 24 h after transduction, and after a further 24 h cells were processed. To delete the Pten gene from cultured Pten flox-flox murine neurons, a modified lentiviral vector was used in which the expression of the RFP-Cre transgene is driven by a human synapsin-1 promoter.31 (link)
32 (link) RFP-Cre lentiviruses were produced by triple cotransfection of 6.5 million HEK293 T cells in 75 cm flasks with 10 µg lentiviral vector, 7.5 µg pHR-CMV 8.9 deltaR packaging vector and 5 µg pCMV VSV-G envelop vector using X-tremeGENE 9 DNA transfection reagent (Roche). The 10 mL of viral supernatant was harvested 48 h after transfection, filtered using 0.22 µ filter (Millipore) and the aliquots were snap frozen in liquid nitrogen for 10 min before storing them at −80°C.