Zoledronate zol

For cultivation according to the R10F protocol MNC at a concentration of 5.0E+05/ml were cultured in 96 U-bottom plates (Greiner bio-one, Frickenhausen, Germany) using standard medium consisting of RPMI 1640 supplemented with 10% fetal bovine serum (FCS), 1% 200mM L-glutamine and 1% penicilline/streptomycin (all from Biochrom, Darmstadt, Germany/Bio&SELL, Feucht, Germany). On day 0, 1 µM Zoledronate (Zol) (Sigma-Aldrich, St. Louis, USA) and 100 U/ml Interleukin 2 (IL-2) (Burton-on-Trent, Great Britain, United Kingdom) were added. From day 7, the cells were harvested twice a week, washed and reseeded at a concentration of 6.0E+05/ml in 96 well plates with fresh medium and 100 U/ml IL-2.

Transduced 293T and MODE-K cells were grown in Complete DMEM supplemented with 1 μg/mL puromycin (Sigma-Aldrich) alone or in combination with 500 ng/mL hygromycin B (Thermo Fisher). Polyethylenimine was from Polysciences. Zoledronate (Zol) was from Sigma-Aldrich.

Zoledronate (Zol) was purchased from Sigma. An anti-OCN antibody was purchased from Abclonal. Anti-IL-1β, anti-TNF-α, and anti-IL-1RA antibodies were acquired from Abcam. Alexa Fluor 488 secondary antibody was purchased from Abcam. IHC-secondary antibodies were bought from ZSGB-BIO. IL-1 RA neutralizing and negative control antibodies were purchased from R&D Systems. IL-1β ELISA kit was purchased from Biolegend. Masson’s trichrome stain kit was obtained from Solarbio. Tartrate-resistant acid phosphatase (TRAP) stain kit was purchased from Sigma-Aldrich. Cell counting kit-8 was purchased from Dojindo.