Rankl

Method for analysis of osteoclast formation was reported previously [15 (link),16 (link),22 (link)]. Briefly, we isolated bone marrow cells from the tibiae of one male mouse (8 week-old) after sacrificed by cervical dislocation under 2% isoflurane anesthesia. For osteoclast formation and quantitative real-time PCR analyses, the bone marrow cells were seeded in 96-well and 24-well plates (cell density: 1×10⁵ cells/cm²) respectively, and cultured in α-MEM (Wako) supplemented with 10% FBS, 1% PS and macrophage colony-stimulating factor (M-CSF, Wako) for 3 days at 37°C for forming bone marrow-derived macrophages (BMMs). For osteoclast formation, the BMMs were cultured in the presence of receptor activator of nuclear factor κB ligand (RANKL, Wako) and M-CSF for an additional 4–5 days at 37°C.
Mouse monocytic Raw264.7 cells (obtained from ATCC) were seeded in 96-well and 24-well plates (cell density: 1×10⁴ cells/cm²) for osteoclast formation and quantitative real-time PCR, respectively. The cells were cultured in α-MEM supplemented with 10% FBS, 1% PS and RANKL for 5 days at 37°C.
The cells were stained with a tartrate-resistant acid phosphatase (TRAP) staining kit (Wako). Number of TRAP-positive multinucleated cells (MNCs) containing three or more nuclei in each well of 96-well plates was counted as osteoclast.

MC3T3-E1 cells (a mouse osteoblastic cell line) were cultured in α-minimum essential medium (MEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS). ATDC5 cells (a mouse chondrosarcoma cell line) were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (DMEM/F-1; Wako) supplemented with 10% FBS. RAW264.7 cells (a mouse monocyte and precursor of macrophage cell line) were cultured in RPMI 1640 medium (Wako) supplemented with 10% FBS. All cells were grown at 37°C in 5% CO₂ and 100% humidity. For induction of differentiation, Osteoblast-Inducer Reagent (Takara Bio, Shiga, Japan) for MC3T3E1 cells, Insulin-Transferrin-Sodium Selenite Supplement (Roche Diagnostics, Mannheim, Germany) for ATDC5 cells, and a combination of 10 ng/ml of recombinant human receptor activator of nuclear factor kappa-B ligand (RANKL; Wako) and 10 ng/mL of recombinant human macrophage colony-stimulating factor (M-CSF; R&D, Minneapolis, MN, USA) for RAW264.7 cells were added to the cultures.

Culture plates were left uncoated or coated with collagen, depending on the experiments. RAW264.7 cells were seeded into 96-well plates (3 × 10³ per well) for 5 d in a growth medium containing 50 ng/mL RANKL (FUJIFILM Wako Pure Chemicals, Osaka, Japan). Opto-RANKc and Opto-RANKm cells were seeded into 96-well plates (3 × 10³ per well) for 5 d and 7 d, with blue light (wavelength = 470 nm) exposure every 2 min, consisting of 12 cycles of 10 ms irradiation, with a 100-ms interval, unless stated otherwise. In 7-d culture, cells were replated into 96-well plates (at a density of 6 × 10⁴ cells per well) after 4 d of culture. The medium was changed every 2 d. For OPG inhibition experiments, OPG (Elabscience Biotechnology, Wuhan, China) was added to the culture medium at a concentration of 500 ng/mL.

Mice pancreatic acinar cell culture was performed as previously described³⁹ . Briefly, the pancreas was removed and minced in small pieces. Then the pancreatic segments were enzymatically digested in 10 mL HBSS (Nacalai Tesque, #17461-05) with 10 mM HEPES (Gibco, #15630-080), 200 U/mL collagenase (Wako), and 0.25 mg/mL trypsin inhibitor (Wako, #202-09221) for 30 min at 37 °C. During incubation, mechanical digestion using serological pipettes was performed every 10 min. After washing, pancreatic acini were resuspended in 7 mL of Waymouth’s medium (Gibco, #11220-035) containing 2.5% fetal bovine serum (HyClone), 10 mM HEPES, 0.25 mg/mL trypsin inhibitor and 25 ng/mL recombinant human epidermal growth factor (PEPROTECH, #315-09), seeded on type I collagen coated six-well plate and cultured at 37 °C overnight.
Cultured pancreatic acini from control of Ptf1a^Cre-ERTM-tdTomato mice was supplemented with 10 μM of 4-hydroxy Tamoxifen (4OHT) (Sigma-Aldrich, #H7904).
For stimulation experiments, 200 ng/mL LPS derived from E. coli (Sigma-Aldrich, #L2630), 20 ng/mL TNF (BioLegend, #575204), 20 ng/mL IL-6 (BioLegend, #575702), or 100 ng/mL RANKL (Wako, #184-01791) were added in medium representatively and cultured at 37 °C overnight. The stimulated pancreatic acini were resuspended in TRIzol and performed qPCR described above.