Solubilization solution stop mix

To evaluate cytotoxicity of FA-3WJ/GDENs, 1 × 10⁴ of HEK293 cells, Raw. 264.7 macrophage and KB cells were seeded in individual 96-well plates 1 day ahead treatment and maintained at 37 °C in 100 µL full medium. FA-3WJ/GDENs and lipofectamine were diluted and added to cells in quadruplicate to a final concentration 80 µg/mL then followed 2-fold series dilution to 40, 20, 10 and 5 µg/mL groups. After 24 hours incubation, 15 µL MTT dye (Promega) were add to individual well then incubate in dark for 4 hours. 100 µL Solubilization solution/Stop Mix (Promega) were add to individual well for dissolving the crystal in dark. Once crystal fully dissolved, OD₅₇₀ were measured by plate reader (BioTek) following manufacture procedures. Untreated groups were used for standardization as 1 and data were process and fit with non-linear regression in Prism 7.

Cells were plated at a density of 1×10⁴ cells/well in 96-well plates. The negative control cells were treated with 1% DMSO. Fifteen microliters of MTT was added to each well after 48 h, and the cells were incubated for an additional 4 h. Subsequently, 100 µl of the solubilization solution/stop mix was added according to the manufacturer’s instructions (Promega, Madison, WI, USA), and the plates were incubated for 60 min. The absorbance was measured at 570 nm and 630 nm using an ELISA reader. The actual counts were calculated by subtracting the absorbance at 570 nm from that at 630 nm. Each assay was performed in triplicate, and the average absorbance was calculated.