The data in Figure 1 is an example of cell-based assay development. HEK 293 cells were transiently transfected with a M1 muscarinic acetylcholine receptor expression plasmid and screened using the FLIPR® Tetra system. The M1 muscarinic receptor is one of five subtypes of muscarinic acetylcholine receptors that are expressed on the surfaces of neurons and other cell types. Muscarinic receptors are frequently used as targets in cell-based assays to identify drugs for treating disorders of the central nervous system. In this experiment, we used a calcium mobilization assay to demonstrate that transiently transfected cells respond robustly in a dose dependent manner to the ligand carbachol. We also show how assay sensitivity can be controlled by varying the concentration of the target expression plasmid that is added to the transfection reaction.
HEK 293H cells were transfected with increasing concentrations of M1 muscarinic receptor plasmid DNA, treated with carbachol, exposed to calcium sensitive dye and assayed on the FLIPR® system. Figure 1A shows that the intensity of the cells' response to carbachol directly correlated with the concentration of plasmid DNA that was added to the transfection reaction. When cells were exposed to increasing concentrations of carbachol, they responded in a dose-dependent manner (Figure 1B). Note that untransfected HEK 293 cells also exhibit an endogenous response to carbachol. However, by increasing the concentration of plasmid DNA in the transfection reaction, the response to carbachol that was mediated through the transiently expressed M1 receptor can be distinguished clearly from the endogenous response. The EC50 value of the transfected cells (0.12 µM) is considerably lower than that of the control cells (7.6 µM), indicating a clear distinction between M1 muscarinic receptor activity and endogenous responses to carbachol.
Researchers must weigh the substantial time and resource commitment of creating stable cell lines against their value when developing new GPCR assays. An additional consideration is the divergence of stable cell lines from the 'normal' biology of cells, including receptor activation and signaling, as evidenced by changes in the performance of stable cell lines in functional assays. In contrast, transient transfection, and more specifically MaxCyte STX electroporation, quickly and reproducibly transfects cells with minimal off target effects and proven performance in downstream GPCR assays such as cAMP regulation and calcium flux assays.
The data in Figure 2 demonstrate the use of large-scale, bulk transfection as a means of over expressing the β2 adrenergic receptor. Two independent large-scale transfections of over 1x109 cells produced high quality, consistent results with cell viability of greater than 97% (data not shown). Electroporation did not significantly affect cell viability or assay quality as the level of propidium iodide exclusion and assay S/B ratios were nearly identical to those of a reference stable cell line over expressing non-GFP linked β2 adrenergic receptor. Importantly, transiently transfected cells performed similarly to the reference cell line in a cAMP assay as demonstrated by comparable isoproterenol EC50 values (Figure 2, table). These results highlight the capacity of the MaxCyte STX to produce functionally relevant cells at the multi-billion cell scale required for HTS.
High content and high throughput campaigns require a large number of cells to perform a single screen. Other transfection technologies require multiple small-scale transfections, re-optimization of transfection protocols and/or bulk usage of costly transfection agents. The MaxCyte STX has the unique scalability to transfect as few as 5 x 105 cells within seconds for assay development and lead optimization or as many as 1 x 1010 cells in less than 30 minutes for library screening and protein production. Transfection quality and performance in downstream functional assays are unaffected by scale-up. Additionally, migration from small-scale to bulk transfection is seamless, requiring no further optimization.
The MaxCyte STX enables bulk transfection of billions of cells in less than 30 minutes using preprogrammed electroporation protocols. Transfected cells can be used in a wide range of assays immediately following electroporation. If more suitable to assay scheduling, transfected cells can also be aliquoted and cryopreserved for future use. MaxCyte has developed several cryopreservation protocols that enable cell preservation while maximizing cell viability and target expression upon thawing.