Mitotracker deep red solution

MDA-MB-231 cells were transfected with LifeAct-GFP to display actin fluorescence. These cells were a kind gift from the Lauffenburger Lab at MIT. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) with 10% fetal bovine serum (Catalog#16000044, Gibco) and 1% penicillin–streptomycin (Life Technologies) are used as culture media. The cells are cultured in an incubator at 37 °C, 5% CO₂. Cells were stained with 500 nM Mitotracker Deep Red Solution (M22426, Invitrogen) for 90 min then washed by DMEM before cell loading. Studies have shown that cell proliferation inhibitors, such as mitomycinC and Paclitaxel, can lead to changes to cell size^{31 (link)} and migratory phenotypes^{21 (link),32 (link)}. Therefore, to maintain physiological relevance, the cells are not treated with any drugs.

Live imaging of the cells treated with compounds was performed on the HeLa cell line. Cells were seeded in Ibidi imaging cell chambers (Ibidi^®, Gräfelfing, Germany) in 500 μL of a medium, with the concentration of 7.5 × 10⁴ cells/well and left in the cell incubator for 48 h (37 °C, 5% CO₂). After two days, the cells were treated with a 1 μM solution of each compound and left in the cell incubator for 60 min to allow the compound to enter the cells. After the incubation, the medium was changed, and 500 μL of 100 nM MitoTracker Deep Red solution (Invitrogen, Molecular Probes) was added to the chambers. Cells were incubated for 20 min (37 °C, 5% CO₂), allowing MitoTracker to enter the cells. After incubation, the medium was replaced with 500 μL of fresh medium. Co-localization of compounds (λ_exc = 405 nm, λ_em = 470–670 nm) and mitochondria (MitoTracker λ_exc = 644 nm, λ_em = 665 nm) was then visualized and confirmed using Leica SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany). Control and untreated cells excluded the presence of foreign fluorescence.