Mouse cytokine array

The mouse cytokine array (RayBio, Norcross, GA, USA) was used to detect cytokines secreted by M2-type TAMs. Briefly, the cytokine antibody-coated membranes were blocked with a blocking buffer for 30 min and incubated with a culture medium of the sorted M2-type TAMs overnight at 4 °C. Then, the membranes were washed and incubated with the biotin-conjugated detection antibody cocktail and the horseradish peroxidase (HRP)-conjugated streptavidin. Finally, the membranes were washed, subjected to chemiluminescence, developed, and photographed.

The cytokine content of the kidneys was measured with a mouse cytokine array that included 97 cytokines (RayBiotech, Norcross, GA, AAM-CYT-3 and AAM-CYT-4). Kidney samples were collected from the control group, PQ-treated group and triple BMCs co-treatment group on days 1, 2, 3 and 6. Kidney supernatant was obtained as follows. First, the kidneys were removed, minced, and homogenized in lysis buffer. The homogenate was then centrifuged at 9,000 × g for 30 min at 4 °C, the pellet was discarded, and the supernatant was stored at –70 °C until use. The cytokine content of the kidney supernatant was performed according to the manufacturer’s instructions. Briefly, the samples were incubated with the membranes for 2 h at room temperature. After washing, the membranes were incubated with biotin-conjugated anti-cytokine primary antibodies, followed by washes and development with HRP-conjugated streptavidin. The membrane was exposed to X-ray film for 30–60 s. The intensities of the cytokine signals were quantified using Image J software (NIH, Bethesda). The densities of the cytokine spots were normalized according to the blank control spot, and a positive spot was used as the internal control to measure the cytokine content of individual samples.

For cytokine array, flushed bone marrow cells were gently washed with serum-free RPMI 1640 (Gibco, Carlsbad, CA) media for 10 min at room temperature, and the media was recovered by centrifugation at 300 g for 5 min. 250 µg of total proteins measured by Bradford assay were applied to a mouse cytokine array (Raybiotech, Norcross, GA), as described previously (16). For western blotting, total tissue samples were solubilized in lysis buffer, and loaded, 20 µg per lane, on 12% SDS-PAGE. Proteins were blotted onto nitrocellulose membrane and probed using anti-TC1 antibody. Anti-β-actin antiserum was used for loading control (Sigma-Aldrich).