Sre0045

Plant leaves were incubated with 100 μg ml⁻¹ luciferase (Sigma, SRE0045) protein solution for 1 h at room temperature, followed by addition of a P. syringae suspension (OD₆₀₀ = 0.2), which was then incubated for 30 min in 23 °C under light before washing three times with sterile ddH₂O. Subsequently, samples were treated with a flash assay buffer containing 20 mM Tricine, 3 mM MgSO₄, 0.1 mM EDTA, 2 mM dithiothreitol, and 500 μM D-luciferin (Sigma, L6882), and fluorescence was monitored using a Zeiss Axiovert 200 M with Leica DFC290 color camera and DFC340 monochrome camera.

Hippocampus were analyzed for luciferase aggregation prevention capacity as previously described (Adelöf et al., 2018). Specifically, right hippocampi were manually grinded in Eppendorf tubes and lysed in extraction buffer (25 mM Tris/HCl pH 7.8, 100 mM NaCl, 5 mM MgCl₂, 1 mM ATP, 1 mM AEBSF, and 5% glycerol), and cell debris was removed by centrifugation at 5000 g for 10 min followed by addition of 1 mM DTT after an aliquot was taken for protein concentration determination with the BCA Protein Assay kit (Pierce, Thermo Fisher Scientific). luciferase (200 nM; SRE0045, Sigma‐Aldrich) was denatured at 42°C in 50 mM Tris (pH 7.6) and 2 mM EDTA, in the presence of 5 µg hippocampal protein extracts (4.5 µg for females 7 months) and aggregation of luciferase was measured as light scattering at 340 nm. Sample wells without protein extract were used as positive control (reference indicating maximum aggregation). Protein samples without luciferase were used as negative control of aggregation and extraction buffer only was considered background, neither of these controls changed over time. The chosen timepoint for analysis was when the positive control (without protein extract) had reached 60%–80% of maximum aggregation. The luciferase aggregation prevention capacity of the hippocampus extracts was calculated as percent non‐aggregated luciferase.

The following optimized composition of the luciferase plug was used for the experiments: 83 μL of Matrigel^® matrix, 10 μg of luciferase enzyme (1 × 10¹¹ units per mg, Sigma-Aldrich SRE0045), 10 mM ATP, 1 mM Mg²⁺ and PBS up to a 100 μL total volume. ATP, Mg²⁺, luciferase and PBS were first premixed in an Eppendorf tube, followed by the addition of ice-cold Matrigel^® matrix. The concentrations of stock solution were as follows: ATP – 100 mM, Mg²⁺ −0.5 M and luciferase - 10 μg/μL. Typically, 1.2 mL of plug solution was prepared and stored at −20 °C until use and 100 μL of the plug solution was used for each mouse injection and experiments in human cadaver. Subcutaneous injection was performed using 1 mL syringe. The size of the plug post injection was ~7 × 7 mm. 500 μL of the plug solution was used for experiments in dogs.