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Application Note 2
Application Note 2: Developing Assays for Screening GPCRs with Transiently Transfected Cells Using the MaxCyte STX Scalable Transfection System.

Application Note 1
Application Note 1: Developing a Luciferase-based NF-κB Reporter Assay with Transiently Transfected HEK 293 Cells.

FR-MIPTEC-Poster
Poster: Rapid Automated Development of Cell-based Assays for Screening GPCRs, Ion Channels and Other Target Molecules Using the MaxCyte STX Scalable Transient Transfection System.

MaxCyte Platform > Technology > Assay Development

MaxCyte scientists have developed an extensive compilation of cell-type specific protocols, thus eliminating the need to optimize electroporation parameters. Assay optimization is a simple, straightforward process accomplished by performing a series of small scale, static electroporations with varying concentrations of DNA. After identifying the DNA concentration that yields the desired balance of transgene expression, DNA toxicity, and performance of cells in downstream applications, users can scale up to flow electroporation for production of large numbers of transfected cells without further optimization. Transfection performance will not be impacted by electroporation scale up.

If users are transfecting cells with other loading agents, such as RNA, proteins, or cell lysates, similar titration studies using the agent of interest are suggested. Other parameters that can affect performance in downstream applications and can be addressed during assay development include post electroporation cell culturing and the timing of cell cryopreservation.


Determining the Optimum DNA Concentration

The primary experiment in the development of transfection protocols is performing a DNA titration followed by analysis of expression levels, cell viability and performance in downstream assays. MaxCyte recommends DNA concentrations between 100µg/mL and 400µg/mL as a starting point. For co-transfection assays, MaxCyte suggests also performing electroporations with several different ratios of the loading agents (multiple plasmids, DNA and siRNA, etc.).

MaxCyte electroporation in itself does not significantly affect cell viability. High concentrations of DNA, however, may decrease cell viability, a phenomenon known as DNA toxicity. Figure 1 represents a basic DNA titration of a GFP expression plasmid in CHO cells which demonstrates the relationship of DNA concentration and cell viability. As expected, cell viability was negatively impacted in a DNA concentration dependent fashion. Transfection efficiency, as measured by the number of GFP positive cells, was 98 to 100% for cells transfected with any of the DNA concentrations. The level of GFP expression (mean fluorescence intensity), however, was DNA concentration dependent. The optimum DNA concentration for a given application is determined by the user to attain the desired balance between transgene expression and cell viability.

Figure 1. DNA titration.
Figure 1. DNA titration. CHO K1 cells were transfected with increasing concentrations of a GFP expression plasmid using small scale, static electroporation. Cells were analyzed 24 hours post electroporation for GFP expression and cell viability.

Downstream Assay Sensitivity is Determined by DNA Concentration

The concentration of DNA used during transfection can affect the sensitivity and dynamic range of downstream assays. This property is highlighted in the reporter gene assay described in Figure 2. In this assay cells were transfected with two concentrations of plasmid DNA that encoded an NF-κB response element linked to a promoter driving expression of firefly luciferase. Following electroporation, cells were plated in 96-well plates at two different cell densities and incubated overnight with 0, 10, or 40ng/mL TNFα. Higher luciferase expression was seen in cells transfected with higher DNA concentration although all cell populations had robust, dose-dependent luciferase expression in response to TNFα stimulation.

Figure 2. Controlling assay sensitivity via DNA concentration and cell plating density.
Figure 2. Controlling assay sensitivity via DNA concentration and cell plating density. HEK 293 cells were transfected using small scale electroporation with 50 µg/mL or 200 μg/mL of plasmid DNA containing tandem copies of the NF-kB response element linked to a minimal promoter driving expression of firefly luciferase (SA Biosciences). Control cells were electroporated in the absence of DNA. Cells were plated in 96-well plates at 5,000 or 20,000 cells/well immediately after electroporation and incubated overnight with 0, 10, or 40 ng/mL of TNFα. Luciferase activity was measured using a Dual-Glo® luciferase assay (Promega). Error bars indicate standard deviations in three replicate wells.

Assay Development for Expression of a Functional GPCR

Figure 3 depicts the development of a cell-based FLIPR assay using HEK 293 cells that are transiently transfected with the M1 muscarinic acetylcholine receptor. A DNA titration was performed, showing concentration dependent expression of the M1 receptor. Cells transiently transfected with 200µg/mL of DNA were stimulated with various agonist concentrations and receptor activation measured via calcium mobilization. Calcium mobilization directly correlated with the concentration of plasmid DNA that was used during electroporation. Note that untransfected HEK 293 cells also exhibited an endogenous response to the agonist. The calculated EC50 value of transfected cells (0.12 µM) is considerably lower than that of the control cells (7.6 µM), indicating a clear distinction between expressed M1 muscarinic receptor activity and endogenous responses to agonist. Overall, these data demonstrate the expression of a functional GPCR with appropriate signaling upon ligand stimulations.

Figure 3. Carbachol-induced calcium mobilization of the M1 muscarinic receptor.
Figure 3. Carbachol-induced calcium mobilization of the M1 muscarinic receptor. A). HEK 293H cells were transfected with increasing concentrations of M1 muscarinic receptor plasmid DNA, treated with 0.1 µM carbachol and assayed via FLIPR®. B). Cells transfected ± 200 µg/mL of M1 muscarinic receptor plasmid were treated with increasing concentrations of carbachol and assessed using FLIPR. Error bars indicate the standard error of the mean derived from measurements in eight replicate wells.

Transgene Expression is DNA Concentration Dependent

MaxCyte transfection was used to develop a potassium channel assay that relied on the over expression of a functional ion channel. CHO K1 cells were electroporated with three concentrations of cDNA encoding the α-subunit of the Kv1.5 ion channel and cell responses measured on the IonWorks Quattro 24 and 48 hours post transfection. High seal resistances and strong current amplitudes in control and DNA transfected cells demonstrate that the electroporation process had no discernable impact on membrane integrity. The percentage of cells expressing currents >0.5 nA correlated with the amount of DNA used in the electroporation reaction.

Figure 4. Transiently transfected CHO K1 cells perform well in automated patch-clamp assays
Figure 4. Transiently transfected CHO K1 cells perform well in automated patch-clamp assays. CHO K1 cells transfected with three concentrations of cDNA encoding the α-subunit of the Kv1.5 ion channel were analyzed 24 and 48 hours post electroporation on the IonWorks® Quattro™, recording in single hole mode. 24 hour evaluation: cell were cultured at 37°C for 6 hours followed by 28°C for 18 hours; 48 hour evaluation: cultured at 37°C for 24 hours followed by 28°C for 24 hours. Data courtesy of BioFocus.