blocking antibody against rat rage  (R&D Systems)

Journal: Clinical and Experimental Immunology

Article Title: High fructose‐induced metabolic changes enhance inflammation in human dendritic cells

doi: 10.1111/cei.13299

Figure Lengend Snippet: Acute exposure (24 h) of dendritic cells (DCs) to high fructose induces advanced glycation end products (AGE) formation and activates the AGE–receptor for advanced glycation end product (RAGE) pathway. DCs cultured in media supplemented with 15 mM glucose/fructose for 24 h or normal media were investigated for the effect on AGE–RAGE pathway. (a) Immunocytochemistry of AGE in DCs ± amino guanidine (AGE blocker); (b) quantification of AGE accumulation in supernatants using enzyme‐linked immunosorbent assay (ELISA). Data are mean ± s.e. of five donors. (c) Quantification of AGE in the DC supernatants by ELISA ± amino guanidine. Data are mean ± s.e. of three donors. (d) Immunocytochemistry of RAGE and (e) RAGE surface expression using flow cytometry. (a,c) Representative of three such experiments with different donors.

Article Snippet: Immunocytochemistry of AGE, RAGE and nuclear factor kappa B (NF‐kB) DCs were seeded onto glass coverslips in 24‐well plates (0·5 × 10 6 cells/ml) in RPMI and treated with 15 mM glucose/fructose for 24 h with or without 1 mM AMG or blocking antibody against RAGE (R&D Systems; clone 176902, 10 µg/ml) added 30 min prior to glucose/fructose treatment.

Techniques: Cell Culture, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Clinical and Experimental Immunology

Article Title: High fructose‐induced metabolic changes enhance inflammation in human dendritic cells

doi: 10.1111/cei.13299

Figure Lengend Snippet: Enhanced activation of nuclear factor kappa b (NF)‐κB signaling pathway in fructose exposed dendritic cells (DCs) and specific role of fructose–advanced glycation end products (AGE). DCs cultured in 15 mM glucose/fructose were investigated for the effect on the NF‐κB pathway, the downstream effector of AGE–receptor for advanced glycation end product (RAGE) pathway activation by assaying (a). Phospho‐Iκbα by flow cytometry; (b) immunocytochemistry of p65 localization in the nucleus; (c) bar graphs depict the effect of AGE–RAGE pathway inhibition on cytokine production by DCs. Glucose–AGE (G–AGE) and fructose–AGE (F–AGE) were prepared in vitro after incubation of the sugars with bovine serum albumin for 6–8 weeks. The specific effect of these AGEs on DCs was evaluated after stimulation for 24 h. (d) Quantification of AGE formation using fluorescence; (f) Effect of G–AGE and F–AGE on phospho‐Iκbα levels using flow cytometry; (f) bar graphs depict the effect of F‐AGE and G‐AGE and unconjugated BSA on cytokines secretion by DCs at 24 h. Data are mean ± of three experiments with different donors.

Article Snippet: Immunocytochemistry of AGE, RAGE and nuclear factor kappa B (NF‐kB) DCs were seeded onto glass coverslips in 24‐well plates (0·5 × 10 6 cells/ml) in RPMI and treated with 15 mM glucose/fructose for 24 h with or without 1 mM AMG or blocking antibody against RAGE (R&D Systems; clone 176902, 10 µg/ml) added 30 min prior to glucose/fructose treatment.

Techniques: Activation Assay, Cell Culture, Flow Cytometry, Immunocytochemistry, Inhibition, In Vitro, Incubation, Fluorescence