Nunc microwell plate

Undifferentiated Lund human mesencephalic (LUHMES) cells^{17 (link)} were expanded on 50 µg/mL poly-l-ornithine (Sigma-Aldrich, St. Louis, MO) coated T75 flasks (EasYFlasks, Nunclon DELTA, VWR, Darmstadt, Germany) in DMEM/F12 (Sigma-Aldrich) supplemented with 1% N2 supplement (Life Technologies, Carlsbad, CA) and 0.04 µg/mL basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, CT).
For experiments, cells were seeded on either T25 flasks or multi-well-plates (Nunc MicroWell plates, Thermo Fisher Scientific, Waltham, MA) sequentially coated with 50 µg/mL poly-l-ornithine (Sigma-Aldrich) and 5 µg/mL bovine fibronectin (Sigma-Aldrich). To induce differentiation of LUHMES cells into dopaminergic neurons, differentiation medium consisting of DMEM/F12 with 1% N2 supplement, 1 µg/mL tetracycline, 0.49 µg/mL dibutyryl cyclic-AMP (Sigma-Aldrich) and 2 ng/mL glial cell-derived neurotrophic factor (GDNF; R&D Systems, Minneapolis, MN) was used. Cell density was kept at 100,000 cells/cm² across all flasks and well plate formats. Cells were kept at all times in standard cell culture conditions at 37 °C, 5% CO₂, and water-saturated air. Cells were routinely tested for mycoplasma contamination.

Lund human mesencephalic cells were cultured as described previously (Höllerhage et al., 2017 ). Briefly, cells were plated in T75 flasks (EasYFlasks, Nunclon DELTA, Thermo Fisher Scientific, Waltham, MA, United States) coated with 50 μg/mL poly-L-ornithine (Sigma-Aldrich, St. Louis, MO, United States) in DMEM/F12 growth medium (Sigma-Aldrich) with 1% N2-supplement (Life Technologies, Carlsbad, CA, United States) and 0.04 μg/mL human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, CT, United States). Multi-well dishes and flasks (Nunc MicroWell plates, Thermo Fisher Scientific, Waltham, MA, United States) were coated with 50 μg/mL poly-L-ornithine (Sigma-Aldrich) at 37°C overnight and washed three times with phosphate-buffered saline (PBS; LifeTechnologies) followed by coating with 5 μg/mL fibronectin (Sigma-Aldrich) for 24 h in the incubator (37°C, 5% CO₂). For experiments, cells were plated at a density of 110,000 cells/cm² in differentiation medium [DMEM/F12 with 1% N2-supplement, 1 μg/mL tetracycline, 0.49 mg/mL dibutyryl cyclic-AMP (Sigma-Aldrich), and 2 ng/mL human glial cell-derived neurotrophic factor (GDNF; R&D Systems, Minneapolis, MN, United States)]. Cells were routinely tested for mycoplasma contamination.

Mixed glial cultures containing astrocytes, pericytes, endothelial cells, and microglia were isolated from human brain tissue as described previously [47 (link)] and used at passage two. Isolated pericyte cultures were generated from these initial mixed glial cultures by subsequent passaging in order to dilute out non-proliferating microglia, astrocytes, and endothelial cells as described previously [48 (link)]. Isolated microglial cultures were generated as described previously [49 (link)]. Cells were harvested using 0.25% trypsin- 1 mM EDTA (Gibco, CA, USA) with mixed glial cultures and microglia cultures also utilising gentle detachment with a cell scraper (Falcon, MA, USA) due to strong microglial attachment. Viability was determined by trypan blue exclusion (Gibco). Mixed glial and pericyte cultures were plated at 15,000 cells/cm² and isolated microglia were plated at 30,000 cells/cm² in Nunc™ microwell plates with Nunclon™ Delta surface (Nunc, Denmark). All cultures were maintained in DMEM/F12 (Gibco), 10% fetal bovine serum (FBS; Moregate, Australia) and 1% penicillin streptomycin glutamine (PSG; Gibco).

The aggregation of R. solanacearum cells was measured in vitro using a slight modification of the polyvinylchloride microtitre plate assay described by O’Toole and Kolter (1998). Briefly, 5‐µL overnight cultures of R. solanacearum grown in ¼ × M63 medium adjusted to OD₆₀₀ = 0.005 were used to inoculate 95 µL of ¼ × M63 medium in the wells of a polyvinylchloride microtitre plate (Nunc MicroWell plate; Thermo Fisher Scientific Inc., Waltham, MA, USA). Tomato apoplast fluid was added to the wells, and the plate was incubated at 30 °C for 24 h without shaking. To quantify the cell aggregation, 25 µL of 1.0% (w/v) crystal violet solution were added to the wells. After a 15‐min incubation, the unbound crystal violet stain was gently removed with a pipette, and the wells were washed with distilled water, 70% ethanol and distilled water again. The remaining crystal violet in each well was solubilized with 100 µL of 100% ethanol, and then quantified by measuring the absorbance at 550 nm. The resulting value was normalized according to the number of cells (OD₅₅₀/OD₆₀₀). This value was considered to represent the relative cell aggregation (Mori et al., 2016). The experiment was repeated three times, with seven technical replicates in each experiment.