Briefly, 50 mg/mL of BSA (Fujifilm Wako, Osaka, Japan) was incubated under sterile conditions with 0.2 M of glycolaldehyde (AGE-3) (Sigma-Aldrich) in 0.2 M of phosphate buffered saline (PBS) (pH 7.4) at 37C for 7 days. purchased from Invitrogen (488: “type”:”entrez-nucleotide”,”attrs”:”text”:”L23351″,”term_id”:”438669″,”term_text”:”L23351″L23351, 594: “type”:”entrez-nucleotide”,”attrs”:”text”:”L23353″,”term_id”:”438673″,”term_text”:”L23353″L23353, Carlsbad, CA, USA). AGE-modified bovine serum albumin (BSA) was prepared as previously described [15]. Briefly, 50 mg/mL of BSA (Fujifilm Wako, Osaka, Japan) was incubated under sterile conditions with 0.2 M of glycolaldehyde (AGE-3) (Sigma-Aldrich) in 0.2 M of phosphate buffered saline (PBS) (pH 7.4) at 37C for 7 days. BSA incubated under the same conditions was used as a control. Excess glycolaldehyde removed from AGE-BSA or BSA solution by dialysis for 2 days at 4C. Cellulose tube 27/32 (UC27-32-100, SEKISUI CHEMICAL, Osaka, Japan) was filled with AGE-BSA or BSA solution and dialysed in 0.02 M of PBS. The endotoxin concentration was 1.2 pg/ml in 100 g/ml of AGE (measured at SRL, Okayama, Japan). The following pharmacological inhibitors and neutralising antibodies were used: sucrose (0.45 M, Fujifilm Wako), chlorpromazine SGC 0946 (20 M, C2481, Tokyo Chemical Industry, Tokyo, Japan), genistein (40 M, G0272, Tokyo Chemical Industry), FPS-ZM1 (0.25C1 M, 553030, Millipore, Burlington, MA, USA), LPS-RS (10 g/mL, tlrl-prslps, InvivoGen, San Diego, CA, USA), and neutralising antibodies against RAGE (polyclonal Goat IgG, final concentration; 20 g/ml, AF1179), LOX-1 (polyclonal Goat IgG, final concentration; 20 g/ml, AF1564), SR-A/CD204 (polyclonal Goat IgG, final concentration; 20 g/ml, AF1797, all R&D Systems, Minneapolis, MN, USA), CD14 (monoclonal Rat IgG2b , clone; 4C1/CD14, final concentration; 20 g/ml, 557896, BD Biosciences, Franklin Lakes, NJ, USA), CD36 (monoclonal Mouse IgG2a , clone; 185-1G2, final concentration; 20 g/ml, MA5-14112, Thermo Fisher Scientific, Waltham, MA, USA), TLR4 (monoclonal Rat IgG2a , clone; MTS510, final concentration; 20 g/ml, 117608, BioLegend, San Diego, CA, USA), TLR5 (monoclonal Mouse IgG2a , clone; 19D759.2, final concentration; 20 g/ml, NBP2-24787, Novus Biologicals, Centennial, CO, USA) and TLR7 (monoclonal Mouse IgG1 , clone; 4G6, final concentration; 20 g/ml, NBP2-27332, Novus Biologicals). Anti-mouse Abs against phycoerythrin (PE)-conjugated SR-A (monoclonal Human IgG1, clone; REA148, final concentration; 4 ng/test, 130-102-328, Miltenyi Biotec, Bergisch Gladbach, Germany), PE-conjugated TLR4 (monoclonal Mouse IgG1 , clone; UT41, final concentration; 50 ng/test, 12-9041-80, Thermo Fisher Scientific), PE-conjugated LOX-1 (monoclonal Mouse IgG2a, clone; 214012, final concentration; 0.5 l/test, FAB1564P, R&D Systems), allophycocyanin (APC)-conjugated CD14 (monoclonal Rat IgG2a , clone; Sa14-2, final concentration; 50 ng/test, 123312, BioLegend), APC-conjugated CD36 (monoclonal Armenian Hamster IgG, clone; HM36, final concentration; 25 ng/test, 102612, BioLegend), or APC-conjugated RAGE (polyclonal Rabbit IgG, final concentration; 50 ng/test, LS-C212626, LifeSpan BioSciences, Seattle, WA, USA) were used for FACS analysis. Isotype negative control antibodies against anti-CD14 antibody (Rat IgG2b , final concentration; 20 g/ml, 553986, BD Biosciences, Franklin Lakes, NJ, USA) and anti-RAGE antibody (Goat IgG, final concentration; 20 g/ml, sc-2028, Santa Cruz Biotechnology, Dallas, TX, USA) were used. Cell culture and stimulation The mouse macrophage cell line RAW264.7 (EC91062702, SGC 0946 DS Pharma Biomedical, Osaka, Japan) was grown in Dulbeccos modified Eagle medium containing 2 mM glutamine and 10% heat-inactivated foetal bovine serum at 37C and 5% CO2. RAW264.7 cells were plated in 24-well CCNF plates at 5C105 cells/well. After RAW264.7 cells had adhered to the plate, they were incubated with different doses of AGEs (2, 20, and 200 g/ml) and LPS (1, 10, 100 ng/mL, and 1 g/mL) for the indicated times according to each experiment. Fluorescent labelling of BSA and AGE-3 using Alexa SGC 0946 Fluor 488 C5 maleimide AGE-3 was fluorescently labelled as described previously [15, 17]. Briefly, each protein was incubated with 20 times the amount of Alexa Fluor 488 C5 maleimide (A10254, Thermo Fisher Scientific) at room temperature for 2 h in PBS. Excess labelling reagent is removed by dialysed with PBS at 4C for 2 days. Total protein concentration was quantified by the Bradford method using a Bradford protein assay kit (Bio-Rad Laboratories, Kidlington, UK). Alexa Fluor 488-labelled compound fluorescence intensity was measured using ARVO MX 1420 (PerkinElmer Japan, Yokohama, Japan) (excitation: 485 nm, emission:.