Osteoblast differentiation medium

Human primary fetal osteoblasts (hFOB) purchased from Cell Applications (San Diego, CA, USA) were allowed to proliferate up to four passages in an osteoblast growth medium (Cell Applications). The cells were detached using a solution of 1% trypsin-EDTA (Wako) and cultured either in 96-well or 24-well plates using an osteoblast differentiation medium (Cell Applications) supplemented with or without 50 or 100 ng/ml of recombinant XCL1 or tumor necrosis factor-α (TNF-α, Peprotech) for 21 days. The cultures were regularly replenished with fresh media every 3 days. Thereafter, the cells in the 96-well plates were washed with PBS containing MgCl2 and CaCl2 (Sigma), fixed using a 10% formalin neutral buffer solution (Wako), and stained either with alizarin red-S or a BCIP/NBT solution (Wako). The optical density (OD) values were read at 490 nm using a Benchmark Plus Microplate Spectrophotometer System (Bio-Rad, Tokyo, Japan). In a separate experiment, differentiated hFOB (1 × 10⁵) were cultured in differentiation medium (Cell Applications) and stimulated with either recombinant human XCL1 or TNF-α (Peprotech) at concentration of 50 ng/ml for 48 h.

Human fetal osteoblasts purchased from Cell Applications (San Diego, CA, USA) were cultured in osteoblast growth medium (Cell Applications). Cells were differentiated in osteoblast differentiation medium (Cell Applications) for 14 days and cultures were regularly replenished with fresh media every 3 days^{40 (link)}. Cells were stimulated with either recombinant human TNF-α (Peprotech) for 48 h in the presence or absence recombinant AnxA1 (R&D Systems).