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Mitochondrial superoxide (SO) analysis by MitoSOX

BS Brian Sacks
HO Halil Onal
RM Rose Martorana
AS Amogh Sehgal
AH Amanda Harvey
CW Catherine Wastella
HA Hafsa Ahmad
ER Erin Ross
AP Adona Pjetergjoka
SP Sachin Prasad
RB Robert Barsotti
LY Lindon H. Young
QC Qian Chen
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MitoSOX (Thermo Fisher Scientific, Waltham, MA) is a novel fluorogenic dye used for detection of SO within the mitochondria of live cells. The lipophilic, positively charged TPP moiety within MitoSOX allows the dye to concentrate within the mitochondrial matrix pending on high mitochondrial membrane potential in live cells. The dye is oxidized by SO to produce red fluorescence. First, cells were treated by Dox (0.1–50 μM) alone, Dox (40 μM) co-treated with MitoQ or SKQ1, or Dox (40 μM) following pretreatment of MitoQ or SKQ1. After 24 h, the medium was removed and cells were washed with PBS. Thereafter, 100 μl MitoSOX (5 μM) reagent was added to each well and incubated for 15 min. Fluorescence intensity was measured after changing the incubation solution to medium at 531 nm/ 593 nm with Fluoroskan Ascent CF. The mitochondrial SO levels were expressed as the ratio between Dox to non-treated control or between pretreatment to Dox, respectively.

Copyright and License information: The Author(s) ©2021
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