U2-OS cells were plated at 200,000 cells per dish on a 35 mm glass bottom dish (MatTek) and cultured overnight. The cells were rinsed once and loaded with 5 µM CellROX® Green Reagent (Catalog # C10444) and 100 nM TMRM in Live Cell Imaging Solution (LCIS, Cat #A14291DJ) for 15 minutes at 37 degrees C. After loading, cells were imaged live with no washing every 20 seconds for seventy minutes on the DeltaVision Core™ inverted microscope at 37 degrees C. Menadione was added to a final concentration of 100 uM after ten minutes of baseline acquisition. For more information please visit: http://products.invitrogen.com/ivgn/product/C10444 The time lapse data shows the loss of signal from TMRM in the red channel as mitochondrial function decreases, concomitant with the onset of a nuclear signal in the green channel as CellRox® Green is oxidized and the reagent migrates to the nucleus to generate a fluorogenic response as the active form of the dye binds to DNA.
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