Gallios instrument

Lamina propria lymphocytes were stained with antibodies for 30 min at 4°C. The following anti-mouse antibodies (all from Biolegend) were used: Alexa Fluor 700-CD45 (Clone 30-F11), APC/Cyanine7-F4/80 (BM8), PerCP-CD11b (M1/70), Brilliant Violet 570-Ly6G (1A8), Brilliant Violet 421-CD86 (GL-1), FITC-CD206 (C068C2), PE-Ly6C (HK1.4), PerCP/Cyanine5.5-CD3 (17A2), BV421-CD4 (GK1.5), PerCP-CD8a (53–6.7) and FITC-B220 (RA3-6B2). All data were acquired on the Gallios instrument (Beckman) and analyzed using FlowJo X (TreeStar) or Beckman analysis software.

Cultured neurons were stained in situ with TMRE (200 nM, Invitrogen Thermo Fisher Scientific Cat:# T-669) for 30 min at 37°C in 5% CO₂–95% air, washed twice in PBS, and released from culture substrates with 0.05% Trypsin-EDTA (Gibco, cat:# 1995647). After centrifugation, the DRG pellets were re-suspended in 200 µl of FACS buffer (PBS 1X, BSA 1X, 2 Mm EDTA). Flow cytometry of TMRE fluorescence was performed on a Gallios instrument (Beckman Coulter) and analyzed using FlowJo10 software. ~3500 events were acquired for each sample. Data are presented as Mean Fluorescence Intensity per experiment. In some studies, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 10 μM for 1 hr) (Sigma, Cat #C2759) was added as a positive control for mitochondrial depolarization.

Flow cytometric analyses and cell sorting were performed on FACSCanto II and FACSAria I instrument, respectively (both from BD Biosciences). On two occasions, cells were additionally analyzed on FACSVerse, LSRFortessa, FACSAria III (all from BD Biosciences), and Gallios instrument (Beckman Coulter). DCV fluorescence was measured in linear mode, whereas the other fluorescence (i.e., DCG, DCO, 7-AAD, DAPI, and antibodies) was measured logarithmically. A minimum of 100.000 events was recorded.
Data were analyzed using FlowJo version 9.6 (TreeStar). Debris was excluded based on forward/side scatter characteristics and doublets were discriminated using the height and width signals of forward scatter. Living (7-AAD- or DAPI-negative) cells were finally analyzed on bivariate dot plots for the indicated parameters.

For the NK cell degranulation assay, 96 well U-bottom plates were coated with 1.5 and 5 µg/ml of anti-NKp46 mAb (clone D2-9A5) and anti-NKp30 (clone 210847) mAb overnight at 4°C. After washing, 10⁵ human primary NK cells were added for each condition along with BrilliantViolet 421-conjugated anti-CD107a mAb (1:400 final dilutions). After 4 h of incubation (37°C, 5% CO₂), cells were washed and stained again with the same anti-CD107a mAb (1:400 final dilution) and anti-NKp46 conjugated with Alexa Fluor 647 (clone 9E2). Percentages of CD107a⁺ cells were assessed from both 9E2-positive and -negative population. The NK-92 cell line was transduced to express NKp46 full-IRES-GFP (NK92-46Full) and the NK-92 cell line expressing NKp46 D2-IRES-GFP (NK92-46D2) were separately co-incubated for 4 h (37°C, 5% CO₂) with HEK293T cells using a E:T ratio of 1:1, 1:2, or 1:5 along with BrilliantViolet 421-conjugated anti-CD107a mAb (1:400 final dilutions). Then, the cells were washed and stained again with BrilliantViolet 421-conjugated anti-CD107a mAb (1:400 final dilutions). NK-92 cells expressing the same level of GFP (correlating with equivalent levels of NKp46 isoform expression) were gated and percentages of CD107a⁺ cells were analyzed. Flow cytometry was done using Gallios instrument (Beckman Coulter) and FACS data were analyzed using FlowJo software (Tree Star, Inc.).

For the in vivo study, mice received subcutaneous injections of MPTP (Sigma-Aldrich, St. Louis, MO, USA) and lipopolysaccharide (LPS) (Sigma-Aldrich), as described in Figure 1A. Mice were sacrificed under deep anaesthesia (CO₂ exposure, Matsuyama Nishi Sanso Company, Matsuyama, Japan), and whole brains were dissected out and subjected to flow cytometry analysis for multicolour immunofluorescence immuno-labelling (Brilliant Violet 570™ anti-mouse/human CD11b Antibody and Pacific Blue™ anti-mouse CD45 Antibody, BioLegend, San Diego, CA, USA) and mitochondrial ROS (MitoROS 520 (AAT Bioquest, Sunnyvale, CA, USA)) as described in earlier studies [24 (link),25 (link)]. For the in vitro study, BV2 cells were exposed with or without LPS (1 μg/mL) and incubated with or without ZNS (100 μM) for 24 h. The cells were then processed for phagocytosis and mitROS assays as described in earlier studies [24 (link),25 (link)]. The Gallios instrument (Beckman-Coulter, Brea, CA, USA) was used to perform flow cytometry of cells, and the results were analysed using FlowJo (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).