Luminescence Detects Efficiency
As in many transfection techniques, electroporation efficiency is easily assessed by transfecting a reporter molecule into the cell. This method is especially effective in difficult-to-transfect cells (e.g., primary cells) where transfection efficiencies can be in the single-digit percentages.
For example, it was recently reported that transient transfection of CHO cells with PerkinElmer’s aequorin platform is a useful method to set up high-throughput screening, said Vincent Dupriez, Ph.D., principal scientist, who added that the Amaxa Nucleofector technology from Lonza has allowed for the transfection in primary cells.
“With the Amaxa Nucleofector technology, DNA is driven directly into the cell’s nucleus and the time from transfection to translation is shorter compared to other technologies, resulting in protein expression that can be observed within a few hours of transfection. This enables us to achieve rapidly a good level of aequorin expression in a large percentage of cells and to take advantage of the high signal-to-background ratio provided by the aequorin technology,” Dr. Dupriez explained.
The data discussed was derived from the transfection of two types of primary cells—human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells from the lung (HMVEC-L) cells. “Aequorin plasmid DNA was transfected into these two cells types, and then we performed sets of aequorin assays,” he added.
“The Amaxa transfection technology now enables using the aequorin technology in primary cells, providing a new pharmacological tool to study endogenous receptors in a natural cellular environment for labs equipped with tabletop-size or larger luminometers,” he continued.
“As an example, subtype-specific histamine receptor agonists and antagonists were used to demonstrate that the histamine receptor subtype 1 is the one responsible for the calcium response to histamine in both cell types tested.
This opens new possibilities for the characterization of the repertoire of receptors functionally present in primary cells and for the characterization of newly developed molecules in physiologically meaningful cellular models,” he concluded.
In her talk at SBS, Nicole Faust, Ph.D., director of R&D at Lonza Cologne, presented data on how primary cells can be used in cell-based assays. She explained that one prerequisite for many cell-based assays is the efficient genetic manipulation of the cells in order to introduce reporter genes or biosensors for second messengers.
“Using the Amaxa Nucleofector technology, we successfully introduced the Ca2+-sensitive photoprotein aequorin into a variety of primary cells and obtained robust signals in response to the appropriate stimuli,” explained Dr. Faust.
“Using these modified primary cells we could obtain pharmacologically relevant dose-response curves for a number of endogenously expressed target proteins.
“So far, use of Ca2+-sensitive photoproteins has been mainly restricted to recombinant cell lines,” she continued. “By transferring this technology to human primary cells, we enable researchers to assay endogenous receptors or other drug targets within their natural context in a relevant cell background.”