Dmem nutrientmixture f12 ham s 1 1

The SH-SY5Y human neuroblastoma
cell line (2 × 10⁵ cells/mL) was cultured in 96-well
tissue microplates (100 μL/well) and allowed to adhere overnight
at 37 °C. The stock of GlcCer (in DMSO) was dissolved in DMEM:Nutrient
mixture F12 (Ham’s) (1:1) (Biological Industries, Israel) at
different concentrations (1–200 μM) for 6 h to generate
GlcCer assemblies (based on ThT results). 1% DMSO was used as the
vehicle. The negative control was prepared as a medium without GlcCer
or DMSO and treated in the same manner. 100 μL of medium with
or without GlcCer aggregates was added to each well. Following incubation
for 24 h at 37 °C, cell viability was evaluated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT) cell proliferation assay kit (Biological Industries, Israel)
according to the manufacturer’s instructions. Briefly, 100
μL of the activation reagent was added to 5 mL of the XTT reagent,
followed by the addition of 50 μL of activated-XTT solution
to each well. After 2 h of incubation at 37 °C, color intensity
was measured using an ELISA microplate reader at 450 and 630 nm. Results
are presented as mean and the standard error of the mean. Each experiment
was repeated at least three times.

The SH-SY5Y and HEK-293 cell lines (2 × 10⁵ cells/mL) were cultured in 96-well tissue microplates (100 µL/well) and allowed to adhere overnight at 37 °C. The conjugate molecules were dissolved in DMEM:nutrient mixture F12 (Ham’s) (1:1) (Biological Industries, Israel) at different concentrations. The negative control was prepared as medium without hybrid molecules and treated in the same manner. 100 µL of medium with or without hybrid molecules were added to each well in triplicate. Following incubation for 24 h at 37 °C, cell viability was evaluated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell proliferation assay kit (Biological Industries, Israel) according to the manufacturer’s instructions. Briefly, 100 µL of activation reagent was added to 5 mL of XTT reagent, followed by the addition of 50 µL of activated-XTT solution to each well. After 2 h incubation at 37 °C, color intensity was measured using an ELISA microplate reader at 450 nm and 630 nm. Results are presented as mean and mean standard error. Each experiment was repeated minimum three times.