Fluorescent Cell-Based Analyses
Dr. Persmark gave an overview of several of Life Technologies’ recently launched reagents for fluorescent cell-based analyses at the “Immunology” meeting. “In cellular imaging, we are focusing efforts on probes and technologies to monitor cell structure and function—some entirely novel and some that draw heavily on our 30-year history, such as Alexa Fluor® dyes. However, the Alexa Fluor dyes are also being used in completely new technology applications, such as the Click-based assays.”
In order to deal with some of the challenges inherent in imaging live cells, Life Technologies has developed BacMam technology, which is based on an insect virus (baculovirus) to efficiently deliver and express genes in mammalian cells.
Premo™ FUCCI Cell Cycle Sensor combines Cdt1 and geminin FP constructs with the BacMam gene delivery system. The Premo sensor is based on the two-color FUCCI sensor developed by Atsushi Miyawaki and colleagues at Riken; depending on the mitotic phase the sensor fluoresces in red or green (or yellow at the G1/S interphase), explained Dr. Persmark.
“Packaging in BacMam particles enables researchers to study the mitotic state of individual cells, including stem cells and neuronal cells that traditionally are challenging to label.”
The BacMam platform 2.0 includes elements that enhance transduction efficiency and expression levels: a pseudotyped capsid protein for more efficient cell entry and genetic elements (enhanced CMV promoter and Woodchuck Post-transcriptional Regulatory Element), that boost expression levels. Baculoviruses do not replicate in mammalian cells and thus have an excellent safety profile and lack cytopathic effects on cells, Dr. Persmark added.
“BacMam reagents have been used in cell-based assays, live-cell imaging, stem cell biology, and many other applications. Owing to the high transduction efficiency and lack of cytotoxicity, it is easy to perform experiments in more biologically relevant cellular models—for example, stem cells, neurons, and primary cells.” Dr. Persmark added that for multicomponent systems that require stoichiometric expression, the transient expression and ability to control expression by simply varying the dose enables analysis and even screening of targets that have proven intractable using stable cell lines.
Dr. Christensen noted that many associate live-cell imaging with high-content analysis in terms of microscopy. “Working down to the sub-cellular level, you can get valuable insight to direct cellular response and intracellular location of functional activity,” she said.
“On the other hand, microscopy lacks a great deal of biological relevance. How a cell responds on a slide can be vastly different from how a cell responds in a living organism. Characterization of cellular responses can easily be accomplished at a whole-animal level through noninvasive optical-imaging techniques.”
Optical-imaging technologies rely on light producing optical reporters such as luciferase and fluorescent proteins, fluorescent dyes, and conjugates. “Genes encoding luciferase and fluorescent proteins can be engineered into cells—for example, cancer cell lines and infectious disease agents or animals such as transgenic mice and rats—to enable them to produce light that can then be visualized through the tissues of a live animal using specialized imaging equipment and software designed and built by the company,” said Dr. Christensen.
“These highly sensitive dual bioluminescence and fluorescence imaging systems allow significantly fewer animals to be used due to the generation of superior data and better biostatistics. Optical imaging enables one to characterize functional processes and responses to therapeutic treatment longitudinally and translationally.”
Dr. Christensen noted that there isn’t any one technology that’s optimal for live-cell imaging. “The combination of microscopic and macroscopic techniques are required to fully understand the complete biology at hand,” she said. Live-cell imaging, however, brings more relevance to biological processes in real time. Identification of an event at a specific time can be easily done on fixed cells, but characterization of an event more often needs real-time monitoring of the biological process.”