Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) assay (ThermoFisher Scientific, Massachusetts, United States) was used to test bEnd.3 cells’ cytotoxicity on the porous membrane of LB2. It is a colorimetric method used to quantify cellular cytotoxicity. Damaged plasma membrane releases lactate dehydrogenase (LDH), a cytosolic enzyme found in several types of cells. LDH catalyzes the conversion of lactate to pyruvate via reduced NAD + to NADH. Diaphorase then uses NADH to reduce the tetrazolium to formazan salt, which can be measured at 490 nm. The amount of formazan is proportional to the amount of LDH released into the medium, indicative of cytotoxicity. To perform the LDH assay, after 7 days, bEnd.3 cells are lysed with a 1 mM PBS/EDTA solution, and then transferred to a 96-well plate. Subsequently, the spontaneous and maximum LDH activity is measured: 50 µL of all the samples are transferred to a new 96-well plate to which 50 µL of reaction mixture will be added. After incubating for 30 min in the dark, 50 µL of stop solution will be added to each well. Non-viable cells convert the tetrazolium salts into formazan red. To measure the absorbance, expressed in optical density (O.D), a spectrophotometric reading was carried out at 490 nm, using a plate reader (Synergy HTX Multi-mode microplate reader). The absorbance of this compound is directly proportional to the amount of LDH released by the cells. Three assays were performed, and for each experimental class, the test was performed in triplicate.