474 scanning fluorescence detector

5.0 × 10⁶ of EC2-1H9 cells or the engineered cells were seeded in T175 culture flask containing MEM-α supplemented with 10% (v/v) dFBS, 3.5 g/L glucose, 1% (v/v) Ab-Am solution, and 20 nM MTX. The culture medium was replaced with serum-free medium (CHO-S-SFM II; Gibco) in 3 days. After 2 days, the culture medium was collected, filtered using 0.45 μm filter (Sartorius, Göttingen, Germany), and dialyzed against phosphate buffer saline (PBS, pH 7.4) at 4 °C, overnight. To purify rhEPO, cultured medium was subjected to EPO Purification Gel (MAIIA Diagnostics, Uppsala, Sweden) according to the manufacturer’s instructions. Purified rhEPO was dialyzed against distilled water at 4 °C, overnight. The concentration was measured using a Quant-iT™ protein assay kit (Invitrogen) and stored at −80 °C until use.
The sialic acid level of the purified rhEPO was examined using the OPD-labeling method as previously described^{26 (link)}. Briefly, sialic acid moieties from the purified rhEPO were released using 0.5 M NaHSO₄ at 80 °C for 20 min. Released sialic acid was labeled with OPD (o-phenylenediamine-2HCl; Sigma) at 80 °C for 40 min. The level of OPD-labeled sialic acid was determined using C18 reversed-phase column (Shim-pack CLC-ODS; Shimadzu, Kyoto, Japan) with 474 scanning fluorescence detector (excitation at 230 nm and emission at 420 nm, Waters).

HPLC was used to assay 5-HIAA. The HPLC was performed based on the method previously described [29] (link) with some modifications. For monoamine analysis, an Agilent HC-C18 analytical column (250 mm×4.6 mm, 5 µm; Agilent, USA) was used. The mobile phase was composed of 20% methanol and 80% aqueous solution, which included 30 mM citric acid, 40 mM sodium acetate, 0.2 mM ethylenediaminetetraacetic acid (EDTA) disodium salt and 0.5 mM octanesulfonic acid sodium salt, at a flow rate of 1.0 ml/min and at pH value of 3.8. The level of 5-HIAA were detected using a Waters 474 scanning fluorescence detector (Waters, USA) with the excitation and emission wavelengths set at 280 nm and 330 nm, respectively.

Quantification of GSH in BAL fluid was performed as reported by Steele et al. (53 (link)). Briefly, GSH was derivatized with 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to the fluorescent GS-ABD analyte. After separation on a C18 reverse-phase high-performance liquid chromatography column (Beckman, 4.6 × 150 mm; particle size, 5 μ), GS-ABD was detected with a Waters 474 scanning fluorescence detector [λ(ex) = 390 nm; λ(em) = 530 nm]. Authentic GSH was used as an external standard. GSSG in the samples was reduced with tris(2-carboxyethyl)phosphine (TCP) to GSH, and then derivatized with ABD-F to obtain the total concentration of thiols ([GSSG] = [GSH]_total – [GSH, prior to reduction with TCP]).