Cytotoxicity Assay for Breast Cancer

Breast cancer cells and MCF-10A cells seeded at 1 × 10⁴ per well in 96-well plates were treated with Mito-ChM or Mito-ChMAc for 24 h, and dead cells were monitored in the presence of 200 nM Sytox Green (Invitrogen). The Sytox method labels the nuclei of dead cells yielding green fluorescence. Fluorescence intensities from the dead cells in 96-well plate were acquired in real time every 5 min for first 4 h, then every 15 min after 4 h using a plate reader (BMG Labtech, Inc.) equipped with atmosphere controller set at 37°C and 5% CO2:95% air using a fluorescence detection with 485 nm excitation and 535 nm emission. To measure the total cell number, all of the samples in each treatment group were permeabilized by adding Triton X 100 (0.065%) in the presence of Sytox Green for 3 h, and maximal fluorescence intensities were taken as 100%. Data are represented as a percentage of dead cells after normalization to total cell number for each group.
The IncuCyte™ Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity as determined by Sytox Green staining at regular cell culture condition [23 (link)]. Additionally, phase-contrast and fluorescent images were automatically collected for each time point to determine morphological cell changes.
For clonogenic assay, MCF-7, MDA-MB-231 and MCF-10A cells were seeded at 300 cells per dish in 6 cm diameter cell culture dishes and treated with Mito-ChM for 4 h. After 7–14 days, the number of colonies formed was determined. The cell survival fractions were calculated according to a published protocol [24 (link)].