Cell counting kit

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.

The cell viability of MDA-MB-231 and NCIH460 cells after bortezomib treatment was determined by using a cell counting kit 8 (Nacalai Tesque) according to the manufacturer's protocol or the trypan blue exclusion test using a hemocytometer.

Subconfluent Vero cells were cultured in a 96-well plate in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, after which they were treated with Stx1a or Stx2a (3 pg/ml) in the absence or presence of a given peptide or MMβA-mono for 72 h at 37 °C. The relative number of living cells was determined using a cell counting kit (Nacalai Tesque, Japan), according to the manufacturer’s instructions.