Sulfosalicylic acid

Durashell AA analysis kit contained internal standard and 17 kinds of amino acid standard, which was purchased from Agela Technologies (Tianjin, China). Oxalic acid, tartaric acid, lactic acid, citric acid, succinic acid, hydrochloric acid (HCl), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄•12H₂O), potassium dihydrogen phosphate (KH₂PO₄), phosphoric acid (H₃PO₄) (all AR grade), and malic acid (BR grade) were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Guanosine 5′‐monophosphate (5′‐GMP) disodium salt hydrate, 5′‐IMP, 5′‐AMP, and cytidine 5′‐monophosphate (5′‐CMP) were purchased from Sigma‐Aldrich (St. Louis, Mo., U.S.A.). Methanol, sulfosalicylic acid, and acetonitrile (ACN) (all HPLC‐grade) were purchased from Fisher Scientific (Shanghai, China). The ultrapure water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). Buffer salt I (0.05 mol/L KH₂PO₄) was prepared by dissolving 6.80 g KH₂PO₄ in 1 L water. Buffer salt II (0.01 mol/L KH₂PO₄) was prepared by dissolving 1.36 g KH₂PO₄ in 1 L water and then adjusted to pH 2.8 by 1 mol/L H₃PO₄.

T. vaginalis strain B7RC2 (ATCC 50167) was cultured in Diamond’s medium supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific), 180 µM ferrous ammonium sulfate (Fisher), 28 µM sulfosalicylic acid (Fisher), and 10% horse serum (Sigma) (complete Diamond’s media) (26 (link)). TVAG_219770 (TvMIF) was overexpressed in Master-Neo-(HA [hemagglutinin])₂ plasmid and transfected as previously described (17 (link)). The plasmid was maintained with 100 µg/ml of G418 (Gibco) for selection. Parasites were cultured at 37°C and passaged daily for 2 weeks or less.

T. vaginalis strain G3, the genome reference strain commonly used in research, isolated from Kent, United Kingdom, in 1963, was used for all experiments in this study (15 (link), 74 (link)). Parasites were cultured in modified Diamond’s medium (75 (link)) supplemented with 10% horse serum, penicillin and streptomycin (Invitrogen), and iron solution composed of ferrous ammonium sulfate and sulfosalicylic acid (Fisher Scientific), as described previously (76 (link)).