Transmembrane Potential

There is a small (approx 70 mV) but measurable difference in electrical potential (the transmembrane potential) between the interior of cells, which is electronegative, and the external solution. It exists because there are concentration gradients of K+, Na+, and Cl" across the cell membrane which arise in part from the impermeability of the plasma membrane to ions and in part from the action of ATP-dependent ion pumps present in the membrane. The transmembrane potential can be monitored by charged dyes that redistribute across the membrane and is a useful indicator of the status of cells. 3,3'-Dihexyloxa-carbocyanine iodide (DiOC6[3]) is a positively charged (cationic) carbocyanine dye that binds readily to negatively charged cells and to negatively charged mitochondria. It can be excited at 488 nm and its fluorescence intensity, which is detectable in FL1, can be used as an indication of the transmembrane potential. Bis(1,3-diethyl barbituric acid trimethine oxonol) or (DiBAC4[3]) (frequently called "bis-oxonol") is a anionic lipophilic dye that can be used instead of DiOC6(3), but as a result of its negative charge, it does not bind to mitochondria and is a better dye for measuring plasma membrane potential. Rhodamine 123 and DiBAC4(3) have been widely used for measuring transmembrane potential in bacteria, but DiBAC4(3) is considered better than DiOC6(3) for use in mammalian cells (3). When measuring the transmembrane potential, the cells must be in protein-free media, the optimum conditions for dye equilibration determined, and hyperpolarizing and depolarizing controls incorporated. Different membrane potentials are created using valincomycin (a potassium ionophore) and buffers of varying K+ concentrations in order to construct a calibration curve that relates fluorescence intensity in an approximate way to the transmembrane potential (24).

Fig. 10. (Continued)

c ch3 ch3

Fig. 10. (Continued)

Fig. 10. The structures of some fluorescent probes that are used for functional studies. (A) The mixed isomers of 2',7'-bis(carboxyethyl)-5-(and -6-)-carboxy-fluorescein (BCECF), a pH-sensitive probe. (B) The mixed isomers of 5- (and 6-)-carboxy-2',7/-diclorodihydrofluorescein diacetate (carboxy-H2DCFDA), a probe for the estimation of intracellular oxidants. (C) The bis-barbituric acid oxonols (DiBAC), which are slow-response membrane-potential dyes: n = 1 in bis(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4[3]), and n = 2 in bis(1,3-dibutylbarbituric acid)pentamethine oxonol DiBAC4(5). (D) The oxa carbocyanines (DiOC), which are slow-response membrane-potential dyes: n = 1 and R = -(CH2)4CH3 in 3,3'-dipentyloxacarbocyanine iodide (DiOC5[3]), and n = 1 and R = -(CH2)5CH3 in 3,3'-dihexyloxacarbocyanine iodide (DiOC6[3]). (E) The cell-permeant Ca2+ indicators Fluo-3, in which R = O", and Fluo-3 AM (Fluo-3 acetoxy-methyl ester), in which R = -OCH2COOCH3. (F) Rhodamine 123, a mitochondrial transmembrane probe.

Fig. 10. The structures of some fluorescent probes that are used for functional studies. (A) The mixed isomers of 2',7'-bis(carboxyethyl)-5-(and -6-)-carboxy-fluorescein (BCECF), a pH-sensitive probe. (B) The mixed isomers of 5- (and 6-)-carboxy-2',7/-diclorodihydrofluorescein diacetate (carboxy-H2DCFDA), a probe for the estimation of intracellular oxidants. (C) The bis-barbituric acid oxonols (DiBAC), which are slow-response membrane-potential dyes: n = 1 in bis(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4[3]), and n = 2 in bis(1,3-dibutylbarbituric acid)pentamethine oxonol DiBAC4(5). (D) The oxa carbocyanines (DiOC), which are slow-response membrane-potential dyes: n = 1 and R = -(CH2)4CH3 in 3,3'-dipentyloxacarbocyanine iodide (DiOC5[3]), and n = 1 and R = -(CH2)5CH3 in 3,3'-dihexyloxacarbocyanine iodide (DiOC6[3]). (E) The cell-permeant Ca2+ indicators Fluo-3, in which R = O", and Fluo-3 AM (Fluo-3 acetoxy-methyl ester), in which R = -OCH2COOCH3. (F) Rhodamine 123, a mitochondrial transmembrane probe.

Table 3

Some Reagent Kits for Assessing Cell Viability

Commercial name Supplier

LIVE/DEAD® Invitrogen

BacLight Bacterial Viability Kit

LIVE/DEAD® Invitrogen

Fixable Violet Dead Cell Stain Kit

LIVE/DEAD® Invitrogen

Viability/ Cytotoxicity Kit

Principle of the assay

SYTO® 9 dye and propidium iodide label live and dead bacteria, respectively

A violet-fluorescent reactive dye (excitable at approx 405 nm) reacts with the surface of live and internal contents of dead cells; subsequent fixation is possible

Esterase action on calcein AM and exclusion of ethidium homodimer I (membrane integrity)

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