Immortal murine mesangial cells and glomerular endothelial cells (Jieqing Biotech, Wuhan, China) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (8 (link), 33 (link)). Immortal murine proximal tubular epithelial cells (kindly provided by Dr. Cheng Pan, Wuhan University, China) were cultured in Dulbecco’s modified Eagle’s medium as described previously (34 (link)).
By using a HiTrap purification kit (GE Healthcare, Port Washington, NY, USA) (8 (link)), anti-dsDNA IgG was purified from the culture supernatants of murine WJ31 hydridoma clone (IgG2a; Jieqing Biotech). Control murine IgG (IgG2a; Jieqing Biotech) was verified by enzyme-linked immunosorbent assay (ELISA) to determine that there was no binding to DNA antigen (data not shown). Cells were starved routinely before the 2-day stimulation of anti-dsDNA IgG or control IgG (2 µg/ml). In some experiments, mesangial cells were stimulated with anti-dsDNA IgG (2 µg/ml) that was premixed withd -form (ALWPPNLHAWVP) or scrambled (PLPHNPWVLAAW) ALW peptide (1 µg/ml). The cell viability of three types of cells was measured using PrestoBlue viability reagent (Life Technologies, Carlsbad, CA, USA).
By using a HiTrap purification kit (GE Healthcare, Port Washington, NY, USA) (8 (link)), anti-dsDNA IgG was purified from the culture supernatants of murine WJ31 hydridoma clone (IgG2a; Jieqing Biotech). Control murine IgG (IgG2a; Jieqing Biotech) was verified by enzyme-linked immunosorbent assay (ELISA) to determine that there was no binding to DNA antigen (data not shown). Cells were starved routinely before the 2-day stimulation of anti-dsDNA IgG or control IgG (2 µg/ml). In some experiments, mesangial cells were stimulated with anti-dsDNA IgG (2 µg/ml) that was premixed with
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