Multi dish culture plate

The cells were similarly seeded on each well of 6 well multi-dish culture plate (Corning Inc., Corning, NY, USA) at a density of 5×10⁴ cells/well and cultured for 24 hr. The medium was changed to OPTI MEM before the complexes prepared at different mixing ratios (5, 10, 20, 40, and 200 pmole MB/μg cationized gelatin) were added. The final concentration of GAPDH MB was 200 nM. After the incubation for 3 hr, the cells were washed with PBS, trypsinized, and collected by centrifugation of 2000 g for 3 min at room temperature. Then, the radioactivity of ¹²⁵I-labeled MB in the cells was evaluated by the gamma counter. On the other hand, the number of cells after the incubation with the complexes was evaluated by the DNA assay. Briefly, the cell lysates prepared by processing with sodium lauryl sulfate (SDS) were mixed with Hoechest solution (Bisbenzimide H33258 Fluorochrome Trihydrochloride DMSO Solution, Nacalai Tesque. Inc., Kyoto, Japan), and then the fluorescent intensity was similarly measured by the plate reader.

Cells were seeded into each well of 96 well multi-dish culture plate (Corning Inc., Corning, NY) at a density of 1 × 10⁴ cells/well and cultured for 24 hr. The medium was changed to OPTI MEM (GIBCO Lifetechnologies Co., Carlsbad, CA, USA), and then cGNS_GAPMB or cGNS_casp3MB (1, 5, 10, 15, and 20 µg/ml) were added to each well. The cell viability was evaluated using a cell counting kit (Nacalai Tesque. Inc., Kyoto, Japan). After the incubation of 3 hr with nanospheres, 10 µl of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) solution was added to each well and further incubated for 1 hr. The absorbance of samples at 450 nm was measured by Microplate Reader. The percentage of cell viability was expressed as 100% for cells without nanospheres incubation.

Cells were seeded to each well of 96 well multi-dish culture plate (Corning Inc., Corning, NY, USA) at a density of 1×10⁴ cells/well and cultured for 24 hr. Then, the medium was changed to the OPTI MEM (Thermo Fisher Scientific Inc., MA, USA), followed by the addition of complexes prepared at different mixing ratios (5, 10, 20, 40, and 200 pmole MB/μg cationized gelatin) to each well. The final concentration of GAPDH MB was 200 nM. After the incubation for 3 hr, 10 μl of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) solution was added to each well and further incubated for 1 hr. The absorbance of samples at 450 nm was measured by the plate reader (SpectraMax i3x, Molecular Devices Japan Co., Ltd., Tokyo, Japan). The percentage of cell viability was expressed as 100% for cells without incubation of complexes.