Application Note

Optimization and Scaled Delivery of Plasmid DNA to Jurkat Cells at 1 Billion Cells/Minute

Updated — July 17, 2026

Abstract

Traditional electroporation systems suffer from several difficulties, including (1) tedious optimization, (2) inflexibility, and (3) poor scaling from R&D to clinical volumes. Here, we use CyteQuest's electroporation platform to optimize and scale transfection of Jurkat cells with plasmid DNA, demonstrating:

  1. Efficient, automated screening of voltage waveforms to optimize transfection
  2. Flexibility in voltage waveform and processing volume
  3. Identical transfection performance when scaling between cell processing speeds

Experimental workflow

Optimize waveform parameters CytoZap Scan Scale processing speed CytoZap Max
Experimental workflow. Waveform parameters were optimized on the CytoZap Scan, then transferred without re-optimization to the CytoZap Max to scale processing speed.

Results

Programmable bipolar rectangular waveforms

Each well was electroporated with a bipolar rectangular waveform defined by three parameters: voltage amplitude (V), pulse duration (t), and frequency (f). Sweeping these parameters across the plate lets the CytoZap Scan identify the combination that best balances GFP expression and cell viability.

Schematic of a bipolar rectangular waveform: a positive pulse of amplitude V and duration t, followed by an inverted pulse of equal magnitude at the half-period, repeating at frequency f.
Figure 1. Schematic of the bipolar rectangular waveform applied during screening. A pulse of amplitude V and duration t is followed by an inverted pulse of equal magnitude and duration at the half-period; the pattern repeats at frequency f. Schematic, not to scale.

Automated waveform optimization of GFP expression in 96-well plate using CytoZap Scan

Jurkat cells were screened with varying electrical waveforms using the CytoZap Scan to optimize GFP expression and cell viability. Optimization was performed rapidly (<10 minutes) using ~20 µL of cells per well, identifying a range of effective waveform parameters. The waveform in well B11 produced the best tradeoff between GFP expression (92%) and cell viability (91%).

96-well plate map of a waveform optimization screen: Jurkat cells transfected with GFP-encoding plasmid DNA on the CytoZap Scan, with each well annotated by its voltage, frequency, and pulse duration and summarizing GFP expression and cell viability per condition. Wells A2 to A4 are near-zero-voltage controls.
Figure 2. Jurkat cells were suspended at 5 million cells/mL, mixed with 200 µg/mL plasmid DNA encoding GFP, and transfected with varying waveforms using the CytoZap Scan. Cell viability (7-AAD dye) and GFP expression were measured 2 days post-transfection using flow cytometry.

Scaling

Optimized on the CytoZap Scan, transferred to the CytoZap Max

Next, the waveform selected in screening was tested on both the CytoZap Scan and CytoZap Max to test scalability. Jurkat cells were transfected in either the Scan (2 million cells/min) or the Max (20 million cells/min) and transfection performance was measured as described in Figure 2. GFP expression was roughly identical in both instruments, while cell viability trended 6–7% higher in the Max compared to the Scan.

Box plots comparing GFP expression and cell viability for Jurkat cells transfected with the selected waveform on the CytoZap Scan versus the CytoZap Max.
Figure 3. Distributions of GFP expression and cell viability for Jurkat cells transfected with either CytoZap Scan or Max using waveform identified in waveform screening.

1 billion cells/minute processing speed

To greatly increase processing speed, we increased our flow rate on the Max (from 4 to 30 mL/min) and cell concentration (from 5 to 33.3 million cells/mL) for a new processing speed of 1 billion cells/min. To maintain performance at faster flow speed, the waveform frequency was increased proportionally with flow speed (12 Hz to 90 Hz), while waveform voltage and duration were unchanged.

These scaled parameters were tested on both the Scan (100 million cells/min) and Max (1 billion cells/min), yielding similar values for GFP expression and viability over two iterations.

InstrumentReplicateGFPViability
Scan194%84%
Max190%93%
Scan293%88%
Max295%93%

Summary

Key findings

  • Fully programmable waveforms. Voltage amplitude, pulse duration, and frequency are all tunable to the cell and cargo for balancing transfection efficiency and cell health.
  • Automated optimization. The CytoZap Scan screens waveforms across a 96-well plate to rapidly optimize GFP expression and cell viability.
  • High expression, healthy cells. Optimized waveforms could achieve >90% GFP expression while preserving >90% cell viability.
  • Seamless scale-up. Performance between the low-volume Scan to high-volume Max was roughly equivalent and permitted operating at high speed: 1 billion cells/min.

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