SH-SY5Y cell viability assays

The viability of SH-SY5Y cells following incubation with glial cell supernatants was evaluated by MTT assays as previously described [19]. Briefly, the viability was determined by adding MTT to the SH-SY5Y cell cultures to reach a final concentration of 1 mg/mL. Following a 1 h incubation at 37 °C, the dark crystals formed were dissolved by adding a SDS/DMF extraction buffer (300 μL, 20 % sodium dodecyl sulfate, 50 % N,N-dimethylformamide, pH 4.7). Subsequently, plates were incubated overnight at 37 °C, and optical densities at 570 nm were measured by transferring 100 μL aliquots to 96-well plates and using a plate reader with a corresponding filter. Data are presented as a percentage of the values obtained from cells incubated in fresh medium only.

The viability of differentiated SH-SY5Y cells was investigated by the lactate dehydrogenase (LDH) release assay [19]. Briefly, cell culture supernatants (100 μl) were pipetted into the wells of 96-well plates, followed by the addition of 15 μl lactate solution (36 mg/ml in PBS) and 15 μl p-iodonitrotetrazolium violet (INT) solution (2 mg/ml in PBS). The enzymatic reaction was started by addition of 15 μl of NAD⁺/diaphorase solution (3 mg/ml NAD⁺; 2.3 mg solid/ml diaphorase). After 1 h, optical densities were measured with a Model 450 microplate reader (Bio-Rad Laboratories, Richmond, CA) using a 490-nm filter. The amount of LDH released was expressed as a percentage of the value obtained in comparative wells where cells were 100 % lysed by 1 % Triton X-100.