In a transwell co-culture system, RAW 264.7 cells (2×105, 4×105, 8×105) seeded on a 0.4 μm Transwell insert (Millipore) were co-cultured with podocytes (4×105) for 48 h in the absence or presence of 25 mM high glucose treatment. The ratios of podocytes (P) to macrophages (M) were 2:1, 2:2 and 2:4, respectively.
In the CM experiments, podocytes (4×105) planted on six well plates were cultured overnight in normal RPMI 1640 media. Then, the cells were washed with PBS three times. After that, normal PRMI 1640 media (control), NC-CM or HG-CMwas added to podocytes for 24 - 72 h. In some experiments, 10μg/ml anti-TNF-α neutralizing antibody (RD, USA), 10μg/ml IgG1 Isotype control (RD, USA), 300μM ROS inhibitors (Tempo, sigma) or 10μM p38MAPK inhibitor (SB203580, RD, USA) was respectively added to cells with CM for 72 h. Besides, 10ng/ml recombinant mouse TNF-α (RD, USA) alone was applied to incubate podocytes for 72 h when necessary.
In the CM experiments, podocytes (4×105) planted on six well plates were cultured overnight in normal RPMI 1640 media. Then, the cells were washed with PBS three times. After that, normal PRMI 1640 media (control), NC-CM or HG-CMwas added to podocytes for 24 - 72 h. In some experiments, 10μg/ml anti-TNF-α neutralizing antibody (RD, USA), 10μg/ml IgG1 Isotype control (RD, USA), 300μM ROS inhibitors (Tempo, sigma) or 10μM p38MAPK inhibitor (SB203580, RD, USA) was respectively added to cells with CM for 72 h. Besides, 10ng/ml recombinant mouse TNF-α (RD, USA) alone was applied to incubate podocytes for 72 h when necessary.