Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Abstract

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration

“…21 It can be measured in intact cells using flow cytometry with MitoSOX, a superoxidespecific fluorogenic probe with a high tropism for mitochondria. 22 Glucose starvation (Figures 4a and b), glycolysis inhibition with iodoacetate ( Figure 4c) and rotenone ( Figure 4d) at the doses used in our previous experiments all independently triggered superoxide production in SiHa cells. More particularly, we documented a B3-fold increase in the mitochondrial superoxide production of (Figure 1c), we did not detect significant changes in the abundance of the corresponding transcripts (Figure 4f).…”

“…MitoSOX Red, a mitochondria-selective fluorescent indicator, was used to specifically detect the superoxide anion 22 according to the manufacturer's instructions. Signals were measured in 50 000 cells on FACSCalibur (BD).…”

The novel indole compound SK228 induces apoptosis and FAK/Paxillin disruption in tumor cell lines and inhibits growth of tumor graft in the nude mouse

“…Several studies have implicated that increased ROS originating from mitochondria plays a role in mitochondrial induced apoptosis, where mitochondrial outer membrane potential collapsed releasing the pro-apoptotic factor, cytochome c, from intermembrane space to cytosol and activating caspases involved in mitochondrial apoptotic pathway. [25][26][27] The Western blot results showed that SK228 caused an increase in the ratio of BAD/Bcl-xL proteins resulting in cytochrome c release for apoptosis initiation. In our previous study, we showed that rotenone could reduce DNA damage, ROS production and apoptosis initiation caused by 3-indole, another indole compound developed by our lab.…”

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

“…Next, to confirm whether a-TOS generates ROS directly in mitochondria we used a mitochondrial-specific probe (MitoSox, Molecular Probes, USA) derived from dihydroethidium and with an attached triphenylphosophonium cation moiety that accumulates specifically in the organelles. 25 Figure 4b shows that the treatment with a-TOS induces ROS generation in mitochondria of NB4 cells. We observed a similar effect with rotenone (an inhibitor of MRC complex I), antimycin A (an inhibitor of complex III) and to a lesser extent with thenoyltrifluoroacetone (an inhibitor of complex II) (Figures 4a and b).…”