Griess assay

Colorimetric assays were used to quantify H₂O₂ and nitrite (NO₂⁻). These were carried out after applying LTP to the specified liquid in the absence of bacteria. Specifically, the potassium titanium(IV) oxalate method was used to measure H₂O₂ as described in [15] (link). Immediately after plasma treatment, of the titanium(IV) solution were mixed with of the plasma-treated liquid and immediately analyzed by UV-V is absorption. H₂O₂ concentrations were determined from the absorbance maxima at 400 nm. The Griess assay (Sigma Aldrich) was used to detect nitrite (NO₂⁻). After treatment, of each sample were mixed with Griess reagent and incubated for 5 min in the dark at room temperature. NO₂⁻ concentrations were determined from the absorbance maxima at 526 nm. Absorbance values were converted to concentrations using calibration curves obtained with H₂O₂ (30 wt%, Fluka) and NaNO₂ ( %, Sigma Aldrich) (Fig. S4 in the supplementary material). Concentrations quoted are after initially correcting for background absorbance and finally for liquid evaporation that occurred during LTP treatment.

Supernatant concentration of TNF-α and interleukin (IL)-6 was determined by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s guidelines (Biolegend, UK). Concentration of nitrite was measured in cell supernatants using the Griess assay (Sigma-Aldrich, UK) as previously described [17 (link), 22 (link)].

Nitric oxide levels released from retinal homogenates were determined by measuring the accumulation of its stable degradation product, nitrite (Griess Assay; Sigma). Retinas were dissected and disrupted manually using dounce homogenizers. Cell suspensions were made in lysis buffer (0.3 mL/retina) as described earlier. Total nitrite levels in retinal supernatants (n = 3 mice per group) were measured in two different dilutions and run in duplicates. Results are reported as µM per milligram of protein.

To detect microglial activation, 1 × 10⁵ BV-2 cells were plated on 12-well dishes in DMEM containing 1% FBS, grown for 20 h, and treated with LPS (1 μg/mL) or α-synuclein fibrils (2.5–5 µM). After 1 day, the release of NO in the media was evaluated by Griess assay according to the manufacturer’s protocol (Sigma-Aldrich). Levels of Iba1 in cells were examined by Western blot as described below using Iba1 antibody (1:500; Wako, Osaka, Japan). In addition, BV-2 cells without or with α-synuclein (2.5 µM) treatment were fixed (4% paraformaldehyde), permeabilized (0.1% Triton X-100), blocked (2% bovine serum albumin), and stained with primary anti-CD68 (1:1000; Cell Signaling, Danvers, MA, USA) or anti-MHCII (1:1000; Invitrogen, Waltham, MA, USA) antibody, followed by secondary antibody conjugated to Cy5 (1:1000; Invitrogen). Nuclei were detected using DAPI (0.1 μg/mL; Sigma-Aldrich). Cells were examined using a Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss Microscopy, Oberkochen, Germany).