*2.6. Cells Culture and MTT Assays*

PC-12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture (100 U/mL penicillin and 100 μg/mL streptomycin). The cytotoxic effects of different selenium concentrations of PSP-SeNPs, SeNPs, and Na2SeO3 on cells were tested using MTT assays [15]. Cells were seeded in a 96-well plate at a density of 1 × <sup>10</sup><sup>4</sup> cells/well and incubated at 37 ◦C in a CO2 incubator (5% CO2 and 95% relative humidity) for 24 h. Then the medium was removed and cells were treated with different concentrations of samples prepared in DMEM with 10% FBS for an additional 24 h. After incubation, 20 μL of MTT (5 mg/mL) was added to each well and incubated at 37 ◦C for 3 h. Then the supernatant was removed and 150 μL of DMSO was added. The absorbance was measured by a microplate reader at 570 nm. The cell viability was calculated by Equation (3).

$$\text{Cell viability } (\%) = \text{OD}\_{\text{sample}} / \text{OD}\_{\text{control}} \times 100 \tag{3}$$

where ODsample is the absorbance of the treated cells and ODcontrol is the absorbance of the control cells.

To determine the protective effect of PSP-SeNPs, SeNPs, and Na2SeO3 on H2O2 induced cell cytotoxicity, cells were pre-incubated with different selenium concentrations of samples prepared in DMEM with 10% FBS for 24 h. After incubation, the medium was removed and cells were washed with PBS. Then cells were treated with a medium

containing 500 μM H2O2 for 12 h. The medium was removed and the cell viability was determined by MTT assay as described above.
