Biology Reference
In-Depth Information
cells for each experimental point) and, in contrast to isolated mitochondria,
the uptake of this lipophilic cation by several intact mammalian cells is a slow
process.
A few years ago, using the lipophilic cation 5,5
0
,6,6
0
-tetrachloro-1,1
0
,3,3
0
-
tetraethylbenzimidazolylcarbocyanine iodide (JC-1), we developed an original
cyto¯uorimetric technique to detect variations in Dc at the single-cell level
(Cossarizza et al., 1993), which has been validated at the single-mitochondrion
level (Cossarizza et al., 1996). This probe allows us to analyze Dc not only ¯ow
cytometry but also by confocal microscopy ( Bednar et al., 1999; Ceruti et al.,
1997; De Maria et al., 1997; Dispersyn et al., 1999; Franceschi et al., 1996;
Guidarelli et al., 1995; Gorman et al., 1997; Salvioli et al., 2000b; Shenker et al.,
1999; Trimmer et al., 2000). JC-1 has an advantage over rhodamines and other
carbocyanines capable of entering selectively into mitochondria, because it re-
versibly changes color from green to orange as membrane potential increases,
i.e., as the mitochondrial membrane becomes more polarized, over values of
about 80±100 mV (Reers et al., 1991; Smiley et al., 1991). This property, dem-
onstrated in Figure 13.2, is due to the reversible formation of JC-1 aggregates,
which causes a shift in emitted light from 530 nm (emission of JC-1 monomeric
form) to 590 nm (emission of J-aggregate), when excited at 490 nm. Both colors
F i g u r e 13.2. Intracellular distribution of JC-1 before and after the depolarization of mitochon-
dria with valinomycin (VAL.). Note that during the staining with this dye, three main events occur:
1) cytoplasmic loading, which is dependent upon plasma membrane potentialÐthe probe remains
in monomeric form, i.e., green; 2) mitochondrial loading, dependent upon DcÐJC-1 forms ag-
gregates and turns orange; and 3) endoplasmic reticulum (ER) loading, that theoretically can take
place after the fall in Dc. In the case of JC-1, aggregates are no longer present when the dye is
released from mitochondria, and the dye becomes green again.
