Cacl2

OGD/R was performed on DIV 10. For OGD/R treatment, OGD media, composed of 130 mM NaCl (Sigma-Aldrich, Saint Louis, USA), 2.5 mM KCl (Sigma-Aldrich), 2.2 mM CaCl₂ (AppliChem GmbH, Darmstadt, Germany), 1.5 mM MgCl₂ x 6H₂O (AppliChem GmbH), 10 mM Hepes (AppliChem GmbH), pH 7.3–7.4, was bubbled with 95% N₂/5% CO₂ for 15 min.
The cells were washed twice with OGD media, then immediately transferred into the Modular Incubator Chamber (Billups-Rothenberg, San Diego, USA) filled with mixed gas containing 95% N₂/5% CO₂ for 15 min at 15–20 L/min. Thereafter, the sealed chamber was incubated at 37°C for 45 min reaching the total time of OGD as 1 h. Neurons in the control group were maintained under normoxic incubation conditions. After OGD, the cells were removed from the chamber, refreshed with previously collected conditioned culture medium and incubated at 37°C in 5% CO₂ for 3 h or 24 h of the reperfusion phase.

Lipolytic activity was assessed through an agar well diffusion assay. More accurately, the medium consisted of 0.5% peptone (LAB M), 0.3% meat extract (LAB M), 0.5% lecithin (Serva, Heidelberg, Germany), 1% tributyrin (Merck, Darmstadt, Germany), and 1.5% agar, with the addition of 2.5 mM CaCl₂ (Applichem) and 5 mM MgSO₄ (Applichem), according to Carrazco-Palafox et al. [29 (link)]. Then, wells were aseptically punched. Overnight yeast and bacterial cultures were centrifuged (12,000× g; 15 min; 4 °C) to obtain CFS. Twenty-five (25) μL of each CFS were added in each well. Incubation took place at 30 °C for 10 days. The presence of a clarification halo around each well was indicative of lipolytic activity.
Strains that exhibited lipolytic activity were subjected to further study. More accurately, overnight culture was centrifuged (12,000× g; 15 min; 4 °C), washed twice with sterile saline, and used to inoculate flasks containing the above medium without agar addition. Incubation took place under shaking (200 rpm) at 30 °C for 21 days. The pH value and total titratable acidity of the samples were determined at days 3, 6, 9, 12, 15, 18 and 21. Uninoculated flasks served as controls. Lipolytic activity was expressed in AU/mL; one arbitrary unit was defined as the amount of enzyme that catalyzed the release of 1 μmol of fatty acids.

The following chemicals were bought from Merck Millipore (Darmstadt, Germany): MgSO₄, ammonium-acetate, citric acid, KCl, Na₂HPO₄, KH₂PO₄, HCl, and EDTA. The following chemicals were bought from Applichem (Darmstadt, Germany): Triton X100, Guanidine, Glutathione, HEPES, CaCl₂, Urea, and NaCl. The following chemicals were purchased from Sigma-Aldrich (Vienna, Austria): Tris-Base, β-mercaptoethanol, L-Arginine, and formic acid. Tween-20 was obtained from Roth (Karlsruhe, Germany), acetonitrile from VWR Chemicals (Radnor, PA, USA) and imidazole from NeoLab Migge (Heidelberg, Germany). The pHLsec plasmids were kindly provided by A. R. Aricescu (Cambridge, UK).