Elisa plate reader at 570

In vitro cell cytotoxicity of DSF/SPC-NSps was evaluated via the MTT assay. 4T1 cells (1.0 × 10⁴ cells/well in 150 μL) were sowed in 96-well plates and incubated 24 h in 5% CO₂ at 37 °C. DSF/SPC-NSps with different concentrations and free DSF solution (DSF in DMSO) were added and incubated for 48 h. Then 20 μL of the MTT solution (5 mg/mL in PBS) were added into each well and incubated for 4 h. The medium was removed and 150 μL of DMSO was added to dissolve the formazan crystals. The maximum absorbance was measured by an ELISA plate reader at 570 nm (Biotek, USA). The cell viability rate was calculated as follows: $$\text{Cell viability rate}\left(\%\right)={\text{OD}}_{\text{t}}/{\text{OD}}_{\text{c}}100\%$$ (OD_t: the mean optical density (OD) of the tested group; OD_c: the mean OD of the control group. Each group’s half-inhibitory concentration (IC₅₀) value was calculated by GraphPad Prism software, Version 7 (GraphPad Software, Inc., USA).