Optimization of the 4D-Nucleofector electroporation code for transfection of adult dorsal root ganglion neurons. (A) Comparison of the 100 μl cuvette and the 20 μl well strip using code DR114 indicated a slightly higher transfection rate with the 100 μl cuvette, but this difference did not reach statistical significance (n = 4–5 wells across two independent experiments). (B) Five 4D-Nucleofector codes were compared using the 20 μl well strips. DR114 consistently resulted in the highest transfection rate, measured 48 h after electroporation (n = 3–5 wells per condition; ANOVA p < 0.001). CU110 did not result in any transfected neurons (not shown). (C) For the four codes that resulted in successful transfection, total neuronal survival rates were examined. CU133 exhibited the most neuronal death, and EM110 produced the highest total neuronal survival rate. Compared to naïve, untransfected cell cultures, electroporated cultures demonstrated a neuronal survival rate of 18–27% (ANOVA p < 0.001). (D) A representative composite image showing approximately 0.75% of the total imaged surface of a 35 mm well and 9% of one 20-frame montage. Beta-III-tubulin labeling (red) indicates neurons, and copGFP labeling (green) identifies transfected cells. Neurons are sufficiently and consistently distributed throughout each well, and the low plating density allows for straightforward neurite tracing and quantification. (E and F) Higher magnification shows neurons with (E) high transgene expression, with copGFP detected in the cell soma and neurites, and (F) moderate expression, with copGFP present only in the cell soma. Scale bar = 300 μm in (D), 50 μm in (E and F). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Influence of serum following neuronal transfection. (A) The omission of FBS and a shorter post-electroporation incubation time did not significantly influence the neuronal transfection efficiency at 48 h. (B) Neuronal survival was significantly affected by both the presence of serum and the incubation time between electroporation and medium change. Omission of FBS from the medium resulted in significant cell death if medium was not changed until 15 h post-electroporation (ANOVA p < 0.001, Fisher's post-hoc analysis; ^* p < 0.05; ^** p < 0.01). However, when the medium was changed 3 h after the electroporation, the omission of serum did not decrease cell survival. (C) Though no significant difference was detected, neurite length was increased on average by 12% after incubation in serum-containing medium for 3 h or 15 h, suggesting that even short-term serum exposure stimulates growth-related mechanisms in adult DRG neurons.

Comparison of transfection efficiency between electroporation devices and programs. Electroporation reactions were conducted in 100 μl vessels using an equal number of DRG cells and a 10 kb plasmid expressing copGFP. Neurons were identified by beta-III-tubulin labeling upon fixation after 48 h in culture. The Lonza 4D-Nucleofector system (white bar) resulted in significantly higher transfection rates compared to all programs used with the Invitrogen Neon device [gray bars; parameters listed are electric potential (V)/pulse width (ms)/number of pulses]. Only the six best electroporation codes chosen from previous pilot screens of 29 Neon programs are shown (symbols indicate individual data points for each condition).

Optimization of DNA amount and cell number for transfecting adult dorsal root ganglion neurons. (A) Altering the amount of DNA and the percent volume of plasmid in the reaction mixture influences the number of transfected neurons at 48 h (n = 3–9 wells across three experiments; ANOVA p < 0.01; black bars). A low amount of DNA (1 μg) resulted in a significantly lower number of copGFP-positive neurons compared to 2 μg DNA comprising the same percent reaction volume (10% of the 20 μl vessel; p < 0.01). Increasing the DNA amount (6 μg comprising 30% of the reaction volume) also resulted in 39%–46% fewer transfected cells compared to reactions with 2 and 3 μg DNA, respectively (p < 0.05). Using 1 μg DNA also lowered transfection efficiencies from values achieved with 2 and 3 μg DNA (p < 0.05; white bars). The condition with 2 μg DNA was set as 100% to normalize across different experiments. (B) Three cell concentrations were tested to determine whether cell numbers would influence the transfection output using 2 μg of DNA. While no significant difference was seen in transfection efficiency at 48 h, lowering the cell number to 1.5 × 10⁵ cells significantly diminished the absolute number of transfected neurons per well disproportionally by more than three-fold (ANOVA p < 0.001). All values are relative to the 2 μg DNA and 3 × 10⁵ cells condition within the same experiment. ^*p < 0.05; ^***p < 0.001.