Anaspec has announced it is releasing a selection of membrane-potential fluorescent probes.
The plasma membrane of a cell typically has a transmembrane potential of approximately -70mV (negative inside) as a consequence of K+, Na+ and Cl- concentration gradients maintained by active transport processes.
Increases and decreases in membrane potential (referred to as membrane hyperpolarisation and depolarisation, respectively) play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signalling and ion-channel gating.
Potentiometric optical probes enable researchers to perform membrane-potential measurements in organelles and in cells that are too small to allow the use of microelectrodes, such as the patch clamp technique.
Moreover, in conjunction with imaging techniques, these probes can map variations in membrane potential across excitable cells and perfused organs with spatial resolution and sampling frequency that are difficult to achieve using microelectrodes.
Potentiometric probes are important tools for studying these processes, as well as for visualising mitochondria (which exhibit transmembrane potentials of approximately -150mV, negative inside matrix), cell-viability assessment and high-throughput screening for new drug candidates.
Potentiometric probes include: the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic and hybrid oxonols and merocyanine 540.
Fluorescent indicators of membrane potential have been broadly classified as either slow (redistribution, or nernstian) or fast (electrochromic) dyes.
There is a difference between these two families of dyes with regard to the mechanism of their response to membrane potential, speed of the response and the range of cellular characteristics.
Fast-response probes (usually styrylpyridinium dyes) operate by means of a change in their electronic structure and consequently their fluorescence properties, in response to a change in the surrounding electric field.
Their optical response is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells and intact brains.
However, the magnitude of their potential-dependent fluorescence change is often small; fast-response probes typically show a 2-10 per cent fluorescence change per 100mV.
Slow-response probes exhibit potential-dependent changes in their transmembrane distribution that are accompanied by a fluorescence change.
The magnitude of their optical responses is much larger than that of fast-response probes (typically a one per cent fluorescence change per mV).
Slow-response probes, which include cationic carbocyanines and rhodamines and anionic oxonols, are suitable for detecting changes in average membrane potentials of non-excitable cells caused by respiratory activity, ion-channel permeability, drug binding and other factors.
A widely used fluorescent dye for measuring transmembrane potential, DiSBAC2(3), has a limitation of poor dynamic range in an assay environment in the absence of serum.
Anaspec has developed a novel membrane-potential sensitive fluorescent dye, HLB021-152, and successfully used it for homogeneous live-cell cAMP assay in serum-containing and serum-free environments.
Upon stimulating the endogenous or heterogenous GPCRs on CNG-channel-cloned HEK 293 cells with agonists, the fluorescent signal of HLB021-152 increased rapidly and had greater dynamic range than DiSBAC2(3).
This new membrane-potential sensitive dye can be formulated for high-throughput screening of GPCR modulators in environments with and without serum.
