Human hb

HEK293 clones expressing human TLR2, TLR9 and S. aureus LTA (LTA-SA) were purchased from InvivoGen (San Diego, California, USA). Human Hb, blasticidin, and hygromycin were from Sigma-Aldrich (St. Louis, MO, USA). Buffy coat from healthy donors was obtained from the Blood Bank, National University Hospital, Singapore. Primary blood cells were incubated in 5% CO₂ at 37 °C in HEPES-buffered RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 2% FBS. HEK293 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Adult Human Hb, which contains 96.5–98.5% HbA1 (α₂β₂ dimer) and 1.5–3.5% HbA2 (α₂δ₂ dimer), has been verified to be in the metHb state by spectrophotometric scanning between 500 and 700 nm (Du et al., 2010 (link)). As the endotoxin concentration in Hb was determined to be 2.86 EU/mg/ml by PyroGene Recombinant Factor C kit (Lonza Inc.), 1 mg/ml of Hb was pre-treated with 5 μg/ml of polymyxin B, which can scavenge 10 EU/ml of endotoxin. The metHb + LTA complex was pre-formed by co-incubating 1 mg/ml metHb with 10 μg/ml LTA for 30 min.

Human Hb: Sigma Chemical Co. (St. Louis, MO, USA); anti-human ADAMTS-5 polyclonal antibody: CEDARLANE Co. (Burlington, Ontario, Canada); Anti-human ADAMTS-8 and -10 polyclonal antibodies: Biorbyt Ltd. (Cambridge, UK); anti-human ADAMTS-9 polyclonal antibody: Sigma; biotinylated link universal streptavidin-HRP secondary antibody and detection system: Dako Co. (Denmark); ELISA kit for ADAMTS-5, −9: Cloud-Clone Corp. (Katy, TX, USA).

Human Hb was bought from Sigma. HPLC-grade Methanol was bought from the Chinese Chemical Reagent Co., Ltd. (Shanghai, China). Water used in this work was Milli-Q water. Wooden tips (toothpicks) were purchased from the local supermarket (Nanchang, China). The length of the wooden tip was cut to ~2.0 cm in this study. All wooden tips were washed with methanol and water and were dried (100 °C) before use. A total of 319 whole blood samples from 319 women (person, their ages were from 21 to 41 and the average age was 28) were collected into EDTA-K2 tube (Shanghai Zhengbang Medical Treatment Technology Co., Ltd., Shanghai, China), including 100 α-thalassemia carriers, 67 β-thalassemia carriers, and 152 healthy samples (without any gene type of thalassemia carriers). All blood samples were collected from Jiangxi Maternal and Child Health Hospital (Nanchang, China). α/β-thalassemia blood samples were firstly confirmed by polymerase chain reaction (PCR) and flow-through hybridization technology analysis (Hybribio Limited, Chaozhou, China) to assign the types of thalassemia carriers; the identification methods followed our previous studies [2 (link),8 (link)].

All experiments were approved by the local Animal Welfare Committee of the Radboudumc (Nijmegen, the Netherlands; DEC 2012-293) in accordance with the guidelines of the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985). Male C57Bl/6 N mice (Charles River) of 8–11 weeks of age were housed under controlled conditions with pulverized standard chow and water ad libitum and randomly assigned to a treatment group.
Human Hb (Sigma-Aldrich, the Netherlands) was dissolved in saline (20 mg/mL) and injected via the tail vein, 250 µL/mouse, weekly for 8 weeks. 24 h urine samples were collected at baseline or immediately after each Hb injection. Kidney tissue was collected in liquid nitrogen and stored at −80 °C or in 4% formalin O/N before imbedding in paraffin.