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Flow Electroporation
Technical Paper: Flow Electroporation Capabilities and Case Studies: Rapid GPCR Screening and Functional Ion Channel Assays.

Advancing Drug Discovery
Poster: Advancing Drug Discovery with the MaxCyte® STX™ Scalable Transient Transfection System: Expression of Intracellular, Membrane-Bound and Secreted Proteins in Physiologically Relevant Cell Lines, Primary Cells and Stem Cells

MaxCyte Platform > Technology > Technology Scalability

High content and high throughput campaigns require a large number of cells to perform a single screen. Large cell numbers are also needed for protein production and use in cell therapy applications. MaxCyte electroporation 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, protein production, and cell therapy. Other transfection technologies require multiple small-scale transfections, re-optimization of transfection protocols and/or bulk usage of costly transfection agents. Transfection quality and cell performance in downstream applications are unaffected by transfection scale up using MaxCyte electroporation. Additionally, migration from small to large scale transfection is seamless, requiring no further assay optimization.

GPCR agonist responses are unchanged by scale up

There are any number of functional and biochemical assay methods for assessing GPCR activation, receptor inhibition and signaling pathway usage. Many of these methods require cellular engineering such as over expression of targets or artificial coupling to specific G alpha subunits. Transient transfection, and more specifically MaxCyte STX scalable electroporation, can quickly and reproducibly transfect large numbers of cells with minimal off target effects and proven performance in downstream GPCR assays such as cAMP regulation and calcium flux assays.

The experiments summarized in Figure 1 were conducted to examine the effects of electroporation run size on the performance of the transfected cells in a functional GPCR assay. HEK cells transfected using both small and large scale MaxCyte electroporation produced comparable concentration-dependent cAMP responses to a natural GPCR ligand. These results highlight the ability of the MaxCyte STX to produce transfected cells at the numbers required for high throughput/high content screening and profiling of GPCR targets. Additionally, the results illustrate the seamlessness of assay scale up as identical electroporation parameters were used an no optimization performed for large scale transfection.

cAMP GPCR Assay
Figure 1. cAMP GPCR Assay: Small vs. Large Scale Electroporation Assay Performance. HEK 293F cells suspended in MaxCyte electroporation buffer (1x108 cells/mL) were mixed with a GPCR expression plasmid (100 µg/mL) and transfected using small or large scale MaxCyte electroporation. Cells were incubated with increasing concentration of the natural ligand and GPCR activity assayed through a cAMP ELISA 18 hours post-electroporation.

MaxCyte scalable electroporation produces similar performance to stable cell lines

To demonstrate the scalability of MaxCyte electroporation, a high content PI3 kinase assay was performed on cells transiently transfected with a plasmid expressing the PI3P binding protein eGFP-2XFYVE using small-scale (SCEP) and large-scale electroporation (LSEP). In addition, a stable cell line expressing the identical reporter protein was tested. No significant differences in IC50 values following exposure to wortmannin, a PI3 kinase inhibitor, were observed among the three cell populations (Figure 2). These results demonstrate the capacity of the MaxCyte STX to produce functionally relevant cells via transient transfection at the multi-billion cell scale. It also highlights the ability to decrease the reliance on stable cell lines as MaxCyte transiently transfected cells produced comparable assay results without the time and cost commitments of creating a stable cell line.

Cells were transfected with a plasmid encoding eGFP
Figure 2: Cells were transfected with a plasmid encoding eGFP expressed as a fusion protein with tandem PI3P binding domains (2XFYVE). Active PI3 kinase leads to localization of GFP to endosomes and visualized as granules. PI3K inhibition leads to redistribution of fluorescence throughout the cytoplasm. Cells transiently transfected via small or large scale electroporation or cells stably expressing eGPF-2XFYVE were incubated for 30 minutes with increasing concentrations of wortmannin. PI3K activity was assessed using high content screening to visualize granule localization.

Similar antagonist profiles following small and large scale electroporation

Jurkat cells were co-transfected with dual reporter plasmids for a nuclear receptor (NR) assay via small and large scale MaxCyte electroporation using identical electroporation protocols to compare assay results. Cells were incubated with a known nuclear receptor antagonist following transfection and a luciferase reporter gene assay performed. The signal-to-background (S/B) ratio and antagonist EC50 values were comparable for cells from both sets of transfected cells, illustrating the scalability and consistency of MaxCyte's transfection process (Figure 3).

Scale up of a dual plasmid nuclear receptor assay
Figure 3. Scale up of a dual plasmid nuclear receptor assay. Jurkat cells were suspended in MaxCyte electroporation buffer with 200 µg/mL of a dual plasmid mixture containing a luciferase reporter plasmid with GAL4 binding sites and an activator plasmid encoding a GAL4 DNA binding domain-nuclear receptor ligand binding domain fusion protein. 4x107 cells were transfected by small scale (static) electroporation in an OC-400 processing assembly; 1.7x109 cells were transfected by large scale (flow) electroporation in a CL-2 processing assembly. Cells were cryopreserved 30 minutes post electroporation. Cells were thawed and immediately plated with antagonist and a luciferase reporter gene assay performed on the following day. Error bars denote standard deviation of 3 replicate wells.