## 2 Materials and Methods
### Scratch Test and NTP Treatment  
At 3 DIV, the cellular monolayer (astrocytes or neurons) was subjected to a focal mechanical disruption using a p200 pipette tip (Figure 1A). Astrocytes and neurons/neurites at the site of this scratch injury were physically disrupted, creating a gap across the culture. In this simple and reproducible model, over time, astrocytes regenerate into the gap, and neurons adjacent to the gap re-grow neurites across the gap. Immediately following mechanical disruption, the media was removed, and cells were washed with phosphate buffered saline (PBS). The PBS was then removed, and cultures were treated with NTP using a nanosecond-pulsed dielectric barrier discharge (nspDBD) system (Figure 1B) [25]. Neurons and astrocytes seeded in 24-well plates (1.6 cm diameter per well) while astrocytes alone were seeded in Transwell inserts (0.7 cm diameter) before subjected to NTP treatment. Two dielectric barrier discharge (DBD) electrodes were used in this study for NTP treatment to maximize the treatment area in wells and Transwell inserts. A flat DBD electrode (1.3 cm diameter) was used to treat monolayers of cells in 24-well plates (Figure 1C) while a pen electrode (0.3 cm diameter) was used to treat cells in Transwell inserts (Figure 1D). Both electrodes used a nanosecond pulser (FPB-20-05 NM, FID GmBH, Burbach, Germany) to generate a high voltage pulse (29 kV, 2 ns rise times, and 20 ns pulse width), and, therefore, the pulse characteristics were the same. While the electrode geometries are different, several studies have demonstrated that the NTP treatment energy is the most crucial effector of biological response with the DBD plasma system [25; 26; 27]. Therefore, both the voltage and current of the discharge in the system was measured and energy per plasma pulse for both electrodes was calculated as previously described [28]: 0.9 mJ/pulse for the flat DBD electrode and 1.6 mJ/pulse for the pen electrode. The DBD electrodes were positioned 1 mm above the cells and plasma was generated in the gap between the electrode and the cells for 10 s. Using an external function generator (TG5011 XT, TTI, Fort Worth, TX, USA), the frequency of plasma pulses was adjusted, between 1 and 15 Hz, to control the energy of treatment. Astrocyte-only and neuron-only cultures were treated with 10, 20, 50, 90, or 140 mJ of plasma; the control group was not treated with plasma following the scratch test (n = 3 for each astrocyte culture group, and n = 11-13 for each neuron culture group). Neurons and astrocytes  
Figure 1: Non-thermal plasma (NTP) was applied to neural cultures using a nanosecond-pulsed dielectric barrier discharge (nspDBD) system for all plasma treatments. (**A**) Primary neurons were plated in 24-well plates, subjected to mechanical disruption via a scratch assay, and treated with NTP 3 days after plating. For the co-culture group, astrocytes cultured in Transwell inserts were added to neuronal cultures at this point, and cell/neurite infiltration into the gap was assessed at 6–10 DIV. (**B**) A nanosecond-pulser was used to generate and deliver high voltage (HV) pulses to the dielectric barrier discharge (DBD) electrode. A function generator was used to control NTP treatment energy by setting the frequency of pulses and the duration of treatment. A z-positioner was used to fix the position of the (**C**) flat DBD electrode or the (**D**) pen DBD electrode 1 mm above the cells treated in 24-well plates or Transwell inserts, respectively. were independently treated in the co-culture group with several combinations of NTP intensities: 0 mJ Neurons/0 mJ Astrocytes, 0 mJ Neurons/20 mJ Astrocytes, 0 mJ Neurons/50 mJ Astrocytes, 20 mJ Neurons/0 mJ Astrocytes, and 50 mJ Neurons/50 mJ Astrocytes. In the co-culture control group, neither astrocytes nor neurons were treated with NTP following the scratch test, but astrocyte-containing inserts were added to the neuronal cultures at the same time point as treated groups (n = 4-6 for each co-culture treatment group).