Genepulser

Electroporation was performed using either a BioRad GenePulser Xcell Electroporation (#1652660) or a Lonza Amaxa 4D Nucleofector (Lonza #AAF-1002B) with the corresponding X unit (Lonza #AAF-1002X). 1 × 10⁶ cells were resuspended in 100 μL of Nucleofection Solution (Lonza) along with 2 μg of guide DNA (pX330 expression plasmid containing a single guide RNA) or 1 μg of pMAX. pMAX was utilized as a nucleofection control containing a sequence for a green fluorescent protein (GFP). Cells were placed in cuvettes with a gap width of 0.4 cm as recommended by the manufacturers for mammalian cells. BioRad cuvettes (BioRad #1652081) were used with the BioRad GenePulser and Lonza cuvettes were used with the Amaxa 4D Nucleofector (Lonza #V4XC-2012). The K562 and Jurkat nucleofection presets were used to deliver the electrical pulse. Successful delivery of plasmid DNA was assessed by analyzing the positive control containing pMAX via flow cytometry 72 h after nucleofection.

Jurkat cells were transfected via electroporation according to a previously outlined protocol.³⁸ (link) After collection via centrifugation at 300 × g, 5 × 10⁵ to 5 × 10⁶ cells were suspended in 100 μL Buffer 1SM (5 mM KCl, 15 mM MgCl₂, 120 mM Na₂HPO₄/NaH₂PO₄, 25 mM sodium succinate, and 25 mM mannitol [pH 7.2]) and briefly (<5 min) incubated with appropriate plasmid DNA (2–5 μg). This solution was transferred into 0.2-cm generic electroporation cuvettes (Bio-Rad Gene Pulser) and immediately electroporated using a Lonza Nucleofector I and X-01 program (X-001 on newer Nucleofector models). Cells were then cultured in pre-warmed recovery media consisting of RPMI with 20% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cell:DNA ratios for optimal gene expression should be empirically determined.