T6399

Cys-alkylation assays were performed using NEM (Sigma-Aldrich, E1271) and Methyl-PEG-Maleimide Reagent (MM(PEG)₂₄) (Thermo Fisher, 22713). NEM was added to Chlamydomonas cultures growing as indicated in each case to a final concentration of 10 mM. After 15 min of NEM incubation, cells were collected by centrifugation (4,000 × g for 4 min), washed in alkylating buffer (50 mM Tris–HCl, pH 7.5; 50 mM NaCl; and 10 mM NEM), and resuspended in a minimal volume of the same buffer. Cells were lysed by 2 cycles of slow freezing to −80 °C followed by thawing at room temperature. The soluble cell extract was separated from the insoluble fraction by centrifugation (15,000 × g for 20 min at 4 °C) and incubated with 10% (w/v) of TCA (Sigma-Aldrich, T6399) for 30 min on ice. Then, samples were centrifugated (15,000 × g for 20 min at 4 °C), and precipitates were washed twice with ice-cold acetone and resuspended in an SDS-containing buffer (50 mM Tris–HCl, pH 7.5; 50 mM NaCl; and 2% [w/v] SDS). Next, samples were incubated with 100 mM DTT_red for 60 min on ice. Finally, samples were again treated with TCA and ice-cold acetone, and precipitates were resuspended in a new buffer (50 mM Tris–HCl, pH 8.0; 50 mM NaCl; 2% [w/v] SDS, 7.5% [v/v] glycerol; and 0.01% bromophenol blue) in the absence or presence of 10 mM MM(PEG)₂₄.

For analyzing protein in the medium, we performed TCA precipitation as previously described [48 ]. Briefly, cell medium was centrifuged at 2,400 g for 5 min to remove cell debris and then subjected to TCA precipitation (up to 10 %) (T6399, Sigma-Aldrich).

Kynurenine was measured in cell culture medium as previously described (38 (link)). Briefly, samples and standards were added to 30% trichloroacetic acid (T6399, Sigma) at a 2:1 ratio, vortexed, then centrifuged at 700 g for 20 min. Supernatant was added to an equal volume of Ehrlich’s reagent (20 mg/mL p-dimethylaminobenzaldehyde (D2004, Sigma) dissolved in glacial acetic acid) and absorbance read at 492 nm (SPECTROstar Nano, BMG Labtech).

BBB permeability was determined using the previously described Evans blue dye (EBD) extravasation method with certain modifications [41 (link)]. Briefly, after 3 d of reperfusion, the rats were anesthetized. Two percent EBD solution (E2129 Sigma-Aldrich) was injected intravenously into each rat at a dose of 100 mg/kg 2 h before sacrifice. In the sacrifice procedure, the rats were deeply anesthetized with 5% isoflurane (3 min) and transcardially perfused with 0.9% saline to remove all intravascular dye, and their brains were quickly removed and coronally sectioned from − 4.3 to + 1.7 mm of the bregma. The selected right (ischemic) cortex was homogenized with 1:2 volume of 50% trichloroacetic acid (T6399 Sigma-Aldrich) for 5 min and then incubated in a dark tube at 4 °C for 24 h. Subsequently, the homogenates were centrifuged at 10000×g for 25 min at room temperature (RT), and the final supernatants were diluted with 1:3 volume of 95% ethanol for the spectrophotometric analysis of EBD (620 nm excitation/680 nm emission wavelengths).

Snap frozen liver tissue (~ 100 mg) was thawed and homogenized in 1 ml water. Next, 125ul of 50% Trichloroacetic acid (TCA, T6399, Sigma Aldrich) was added to the homogenate and incubated on ice for 20 min. After incubation, samples were centrifuged at 1000 RPM for 5 min at 4 °C. The pellets were hydrolyzed in 12 N HCl overnight at 110 °C. The dried pellets were reconstituted in water and incubated for 6 hrs at room temperature, and incubated in chloramine T solution (1.4% chloramine T, 402869, Sigma Aldrich in 0.5 M Sodium Acetate, S2889, Sigma Aldrich and 10% isopropanol, I9516, Sigma Aldrich) for 20 min at room temperature. Finally, 500ul of Ehrlichs solution (1 M p-Dimethylaminobenzaldehyde, 156477, Sigma Aldrich in 70% isopropanol and 30% perchloric acid, 311421, Sigma Aldrich) was added for 40 min at 65 °C. The absorbance of each sample was measured at 550 nm.

Cell growth was measured by a sulforhodamine B (SRB) colorimetric assay^{54 (link)}. Cells were seeded in triplicate at a predetermined density (500–2000 cells per well) in 96-well plates. Cells were fixed in place with 100 μl 10% trichloroacetic acid (Sigma, T6399) at 4 °C for 1 h. After washing and air drying, cells were then stained with 50 μl of 0.057% (wt/vol) SRB (Sigma, S1402, dissolved in 1% acetic acid) for 30 min. Unbound dye was washed away once with water and three times in 1% acetic acid. The cells were allowed to air dry before the dye was solubilized by adding 200 μl of 10 mM Tris base solution (pH 10.5). Conversion of colored end product was measured by optical density at 510 nm in a TECAN Infinite M200PRO plate reader.