Human il6 antibody

IL-6 was quantified from the cell supernatant of the challenged cells by ELISA. Briefly, cells were seeded at a density of 100 × 10⁴ cells in a 35-mm culture plate and grown to confluence. The confluent monolayer was washed twice and kept in media supplemented with 1% ITS. Then incubation with TLR3 ligand and MyD88 inhibitor was performed for 36 h. Two replicates per sample were run in each independent experiment. At the end of incubation, the condition media were collected, and the quantity of the secretory IL-6 was estimated using a commercially available ELISA kit (R&D Systems, DY 206-05) with human IL6 antibody (R&D Systems, DY 008).

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.

A tissue array of multiple organ tumors (MC6163) was purchased from US Biomax (Rockville, MD, USA). The paraffin‐embedded array was deparaffinized and rehydrated followed by antigen retrieval using 10 mm sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by 3.0% hydrogen peroxide. The array was then blocked with Blocking One reagent (Nacalai Tesque) and incubated with anti‐ZEB1 antibody (NBP1‐05987; Novus Biologicals) and human IL‐6 antibody (R&D systems). Anti‐rabbit Alexa488 and anti‐goat Alexa594 were used as the secondary antibodies. The array was mounted with DAPI‐containing mounting medium. Tile scanning was performed using the Leica DMI6000 B inverted microscope with adaptive focus control (Leica Microsystems, Wetzlar, Germany). The signal intensity of each array spot was scored by two researchers (A.K. and Y.T.).