In many areas of life science—from basic biology through biotechnology and pharmaceuticals—researchers want to work on living cells. Moreover, they want to track interactions in these cells, such as how proteins interact and where. That often requires fluorescent markers, and scientists keep making new ones. In the July 18, 2007, issue of the Journal of the American Chemical Society, scientists from the Pacific Northwest National Laboratory, Richland, Wash., reported on a new red fluorescent probe for live cells. Beyond the dyes, some of the most recent commercial advances in this area involve related tools—things that make the markers work better.
For one thing, scientists need ways to see fluorescent markers. That requires filters that pick up the various wavelengths of light. Earlier this year, Omega Optical, Brattleboro, Vt., brought out 15 new filter sets for fluorescent proteins. These sets work with a variety of markers, including some made specifically for live-cell applications, such as Clontech’s (Mountain View, Calif.) Living Colors Fluorescent Proteins.
Beyond being able to see fluorescent markers, scientists also struggle with photobleaching. To reduce that problem, PerkinElmer, Waltham, Mass., developed its UltraVIEW PhotoKinesis Accessory. Adding this to the UltraVIEW system limits the illumination to an area of interest. As a result, a researcher can intentionally activate a specific area or even illuminate an area over time to intentionally photobleach it—all in a controlled size and even shape of illumination. This accessory even includes technology that tracks moving regions.
The HCS Reagent kits from Cellomics, a part of Thermo Fisher Scientific (Pittsburgh, Pa.), also stay on the move. These kits support high-content analysis of a range of parameters, including recent additions for apoptosis and cytotoxicity. Moreover, these kits work with both fluorescence-based platforms for high-content screening and conventional fluorescent microscopes.
Advances for applying fluorescence to live-cell experiments also come in complete systems, such as PerkinElmer’s new Opera LX. PerkinElmer provided this new option to get researchers into this technology at a lower cost, while still being able to upgrade to the capabilities of the higher-end Opera QEHS. As product literature points out, “HCS allows researchers to study how drugs, proteins, and other biological materials affect cell viability, gene expression, or signaling pathways by using automated microscopes to take images of cellular models where proteins of interest are detected using fluorescent reporter molecules or antibodies.” With the Opera LX, live-cell applications can also be used.
Even with so many improved tools, researchers continue to advance live-cell techniques. For example, Xiaowei Zhuang of Harvard University and her colleagues developed a technique called stochastic optical reconstruction microscopy (STORM). With this approach, a researcher combines a series of images—with various fluorescent probes turned on or off—to locate proteins in the cell. Such switchable probes will surely play a role in various live-cell experiments ahead. (See the August, 2007 issue for more.)
|Watching Neuronal Uptake
Many diseases and treatments—especially for depression or neurodegenerative diseases—depend on neurotransmitters. Following these molecules, though, creates a challenge and typically requires the use of radioactive labels. Recently, Molecular Devices, Sunnyvale, Calif., introduced a live-cell assay that measures the uptake of three neurotransmitters: dopamine, norepinephrine, and serotonin. Like a traditional radioassay, the Neurotransmitter Transporter Uptake Assay Kit can deliver an endpoint measurement of neurotransmitter uptake, but it does that in high throughput. In addition, this kit can be used to measure the kinetics of neurotransmitter uptake. Either way, researchers can use 96- or 384-well plates.
As with other cell-based assays from Molecular Devices, the neurotransmitter assay relies on a masking dye licensed from Bayer. This dye reduces extracellular fluorescence, which improves the signal-to-noise ratio for the proprietary dye used for the neurotransmitter uptake. A researcher simply incubates cells in these dyes and then reads the uptake results with a bottom-read mode fluorescence microplate reader. Calibration tests of this kit also produced reliable results. According to Molecular Devices, “There was good correlation between the IC50 values with this assay versus expected literature values.”
About the Author
Mike May, PhD, is a publishing consultant for science and technology based in Minnesota.
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 10, October, 2007, pp. xx-xx.
Filed Under: Drug Discovery