Fluorescence detector

FSEC experiments were performed with either cell lysate or purified proteins. In all experiments, a high-performance liquid chromatography (HPLC) with autosampler (Shimadzu) was connected to a Superose 6 Increase 10/300 GL column (Cytiva) followed by a fluorescence detector (Shimadzu).
For lysate experiments, HEK293 cells expressing mGITR constructs fused to EGFP were resuspended in buffer containing 20 mM tris (pH 8.0), 300 mM NaCl, and 50 mM DDM and extracted at 4°C with gentle rocking for 1 hour. Lysate was clarified by ultracentrifugation before injecting into the HPLC running buffer containing 20 mM tris (pH 8.0), 300 mM NaCl, and 0.5 mM DDM. EGFP fluorescence was monitored using excitation and emission wavelengths of 488 and 509 nm, respectively.
For experiments with purified proteins, samples were run on the HPLC buffer containing 20 mM tris (pH 8.0), 300 mM NaCl, and 0.5 mM DDM. To monitor EGFP fluorescence, excitation and emission wavelengths of 488 and 509 nm were used, respectively. To monitor tryptophan fluorescence, excitation and emission wavelengths of 280 and 335 nm were used, respectively.

Concentrations of serum amino acids, except tryptophan, were determined as previously described (Espe et al., 2014 (link)). In brief, serum samples were oxidized using a hydrogen peroxide solution (containing phenolic formic acid) at 0 °C for 16 h, and sodium pyrosulfite was added after the reaction to decompose the excess peroxyformic acid. Subsequently, 6 mol/L HCl solution was added to the oxidized samples and hydrolyzed at 110 °C for 23 h to release free amino acids. The concentrations of free amino acids were then determined on an amino acid analyzer (Biochrom, Version 30, Biochrom Ltd., Cambridge, UK) equipped with an ion-exchange column. Tryptophan was determined by a high-performance liquid chromatography (HPLC; Shimadzu, Tokyo, Japan). In brief, samples were saponified under alkaline conditions with barium hydroxide solution in the absence of air at 110 °C for 20 h in an autoclave. After adjusting the hydrolysate pH to 3.0, the tryptophan was separated by reversed-phase chromatography RP-18 on a HPLC column, and the chromatograms were integrated using Labsolutions software with a fluorescence detector (Shimadzu, Tokyo, Japan).

An aliquot (2 μg) of CS was digested with CSase ABC (10 mIU) at 37 °C for 2 h in 50 mM Tris-HCl, 50 mM NaAc, PH 8.0. The digest was labeled with 2-AB [32 (link)] glycosaminoglycan-derived oligosaccharides labeled with a fluorophore 2-aminobeand subjected to anion-exchange HPLC on a YMC-Pack PA-G column eluted with a linear gradient from 16 to 474 mM NaH₂PO₄ over a period of 60 min being monitored at Ex 330 nm and Em 420 nm using a fluorescence detector (Shimadzu Co., Ltd., Kyoto, Japan) [30 (link)].

The structural composition of CS/DS and HS/HEP was done via disaccharide characterization with strong anion chromatography (SAX) using high-pressure liquid chromatography (HPLC) at the Complex Carbohydrate Research Center (CCRC) at the University of Georgia (Supplementary Figures S1 and S2). Given the limited amount of material obtained from the mouse corneas, samples were subjected to a single instrumental run, as previously shown [124 (link),125 (link)]. Briefly, SAX-HPLC analysis was carried out using a 4.6 × 250 mm analytical column (Waters Spherisorb) with 5 μm particle size at 25 °C using an Agilent system. Disaccharides were eluted with a gradient from 97% of 2.5 mM sodium phosphate, pH 3.5 and 3% of 2.5 mM sodium phosphate, 1.2 M NaCl, pH 3.5 to 100% of 2.5 mM sodium phosphate, 1.2 M NaCl, pH 3.5 over a 55-min period at 1 mL/min. Disaccharides were detected by post-column derivatization by combining the eluent at a 1:1 ratio with 0.25 M NaOH and 1% 2-cyanoacetamide pumped at a flow rate of 0.5 mL/min from a binary HPLC pump. The mixture was heated to 120 °C in a 10-m reaction coil, cooled in a 50-cm cooling coil and directed into a Shimadzu fluorescence detector (λex = 346 nm, λem = 410). Elution profiles were compared to that of commercial standard disaccharides (Dextra Laboratories).