Il 6 antibody

Peripheral blood mononuclear cells (PBMCs) were isolated from a buffy coat (Sanquin) as described previously [26 (link)]. Buffy coats were obtained from blood donated by healthy blood donors at Sanquin Blood Supply, Amsterdam, The Netherlands. PBMCs were seeded at 5x10⁵ cells/well of 96 well plates or on bovine bone slices in DMEM containing 10% FCS, antibiotics, and control-CM, CXCL8-CM from 200 pg/ml CXCL8 treatment, CCL20-CM from 500 pg/ml CCL20 treatment, CXCL8+CCL20-CM, and TNF-α-CM (ratio DMEM:CM = 1:1 (v/v)). Twenty-five ng/ml recombinant human M-CSF (R&D Systems, Minneapolis, MN) was added to the cells from day 1 to day 3. Ten ng/ml M-CSF and 4 ng/ml human RANKL (Peprotech, London, UK) were added from day 3 to day 21. To similar cultures, 0.15 μg/ml human IL-6 antibody (Clone #6708, R&D Systems) was added and IgG isotype control (Clone #11711, R&D Systems) was used as control for IL-6 antibody. PBMCs were also cultured with DMEM containing 10% FCS, antibiotics, and either CXCL8 (200 pg/ml) or CCL20 (500 pg/ml). After 3 weeks, cells were fixed in 4% formaldehyde, and stained for tartrate-resistant acid phosphatase (TRACP; Sigma). Nuclei were visualized by 4’,6-diamidino-2-phenylindole (DAPI) staining. Osteoclastogenesis was assessed by counting the number of TRACP-positive osteoclasts containing >3 nuclei per cell on 10 pre-determined microscopic fields in the each well.

When confluence was reached, preadipocytes (n = 6 wells per group) were cultured for 24 h in preadipocyte growth medium, which was replaced with 25% MacCM (diluted in RPMI-1640) to elicit inflammatory responses, together with IL-6 antibody (300, 350 and 450 ng/ml) or IL-1β antibody (15 μg/ml as previously established [18 (link)]) (R&D Systems, UK) to block IL-6 or IL-1β action by neutralization for a further 24 h before cell and supernatant collection. Isotype control mouse IgG at the same concentrations confirmed that non-specific binding did not block inflammatory responses (data not shown). The control received a mock treatment of 25% preadipocyte growth medium (diluted in RPMI-1640).

An immortalized human podocyte cell line was cultured and maintained as described previously 5. Briefly, cells were routinely cultured in RPMI1640 medium supplemented with 10% foetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. Firstly, cells were incubated at 33°C for proliferation, and after reached at 70% confluence, the cells were transferred to 37°C for 2 weeks to allow differentiation. Differentiated podocytes were exposed to media containing high glucose (HG, final glucose concentration 30 mmol/l) or 19.9 mmol/l mannitol as osmotic control.
After individual pre‐treatment with gp130 antibody (2 μg/ml, R&D Systems, Minneapolis, MN, USA), IL‐6 antibody (1 μg/ml, R&D Systems, Minneapolis, MN, USA), recombinant sgp130 (1 μg/ml, R&D Systems, Minneapolis, MN, USA), recombinant human IL‐6 (20 ng/ml, Peprotech, Rocky Hill, NJ, USA) or complex of IL‐6 and recombinant human soluble IL‐6R protein (30 ng/ml, R&D Systems, Minneapolis, MN, USA), podocytes were exposed to HG or osmotic control for 24 hrs.

Cell lysates were prepared in radioimmunoprecipitation (RIPA) buffer with protease inhibitor phenylmethanesulfonyl fluoride (PMSF) and Halt™ phosphatase inhibitor cocktail (Roche, Switzerland). The protein concentration was determined with BCA Protein Assay Kit. The samples were separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies used include rabbit anti-TRAF6 polyclonal antibody (pAb), rabbit anti-NF-κB p65 pAb, rabbit anti-phospho-NF-κB p65 pAb, rabbit anti-Flag pAb (SIGMA, USA), mouse anti-GST monoclonal antibody (mAb), mouse anti-GFP mAb and mouse anti-Flag mAb (CWBIO, China). After five washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (or mouse) IgG (Vazyme, China) secondary antibody for 2 h at room temperature. After another five washes, the signal was detected using an image analysis system (Bio-Rad, USA). The secreted protein levels of IFN-β and IL-6 in cell culture supernatant were determined with IFN-β antibody (LSBio) and IL-6 antibody (R & D Systems), respectively. All samples were in triplicate and read at 450 nm using a Multiskan FC Microplate Photometer (Thermo, USA).