Isotype control igg

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.

CM from CAFs was incubated for 2 h with 10 μg/ml mouse anti-mouse WISP-1 neutralizing antibody (R&D Systems) or 10 μg/ml IgG isotype control (R&D Systems). The neutralization efficiency of the anti-WISP-1 antibody was tested by WISP1 ELISA before use.

Whole blood from human healthy donors was obtained at the Blood Donor Center (Karolinska University Hospital, Solna) and neutrophils were isolated using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, 8 ml of whole blood per donor were incubated (5 min, room temperature) with antibody-conjugated magnetic beads that recognize surface antigens on all cell types present in human blood except neutrophils (negative selection). Samples were then subjected to a magnetic field (15 min) and non-target cells bound to the tube walls. Unbound neutrophils in plasma were pipetted into a separate tube and residual erythrocytes were removed via magnetic separation as described above using the MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec). Neutrophils were separated from plasma by centrifugation (400 RCF, 5 min), and resuspended in RPMI-1640 containing 0.2% penicillin-streptomycin (both from Gibco, Waltham, MA) and 10% donor-specific plasma. Resuspended neutrophils were seeded in 24-well plates (1×10⁶ cells/well) and stimulated (6 or 18 h, 37°C, 5% CO₂) in duplicate with LPS (E. coli 0127:B8, Sigma-Aldrich, Burlington, MA), recombinant human IL-26 dimer protein, human anti-IL-26 antibody, IgG isotype control (all from R&D Systems, Minneapolis, MN), or PBS (control).

The expression levels of MICA on the cell surface were determined using flow cytometry, as described previously [23 (link)]. Briefly, cells were hybridized with anti-MICA (1:500; R&D Systems, Minneapolis, MN, USA) and isotype control IgG (1:500; R&D Systems) in 5% BSA/1% sodium azide/PBS for 1 h at 4°C. After washing, cells were incubated with goat anti-mouse Alexa 488 (1:1,000; Molecular Probes, Eugene, OR, USA) for 30 min. Flow cytometry was performed and the data analyzed using Guava Easy Cyte Plus (GE Healthcare, Little Chalfont, UK).

Anti-WISP1 monoclonal antibody, isotype control IgG and recombinant WISP1 protein were purchased from R&D Systems (Minneapolis, MN, USA).