Osteoclast differentiation and PIT formation assay

After sacrifice, the long bones were excised and all the muscle was removed. The epiphyses were cut off and the bone marrow was flushed out using a syringe with a 27 gauge needle with α-MEM containing 1 % Penicillin/Streptomycin/10 % FBS. The cells were separated by using a pipette and left in the incubator overnight. The day after, only the floating cells were collected, centrifuged, and cultured with α-MEM containing 1 % P/S, 10 % FBS, and 30 ng/ml of MCSF. After 3 days, the cells that were not attached were removed and the media was replaced with α-MEM containing RANKL 50 ng/ml. The media with new RANKL was changed every 2 days. Osteoclast differentiation was tested by using TRAP staining, as previously described [6] and by gene expression analysis by using RT-PCR. To evaluate the expression of macrophages and osteoclast markers in A2BRKO and WT osteoclast, RNA was extracted after 7 days of differentiation. RNA extraction was performed using RNeasy Mini Kit (Qiagen, Invitrogen) and QIAshredder columns (Qiagen, Invitrogen), following the manufacturer’s protocol. RNA reverse transcription was performed using the MuLV Reverse Transcriptase PCR Kit (Applied Biosystems). After RNA reverse transcription to cDNA, real-time PCR reactions were performed for a relative quantification of ADGRE and Cathepsin K performed on a Stratagene Mx3005P (Agilent Technologies, CA, USA) with Brilliant SYBR Green Kit QPCR Master Mix (Stratagene, Agilent Technologies, CA, USA), according to the manufacturer’s protocol.

Osteoclast functionality was tested using the pit formation in dentin assay. For this assay, cells are cultured and differentiated on dentin slices. After 7 days, cells were removed by washing the slices with 50 % bleach for 10 min and the dentin discs were stained with 1 % Toluidine blue for 5 min. Resorption pits are visualized by light microscopy and resorption area was quantified using ImageJ software (NIH, Bethesda, MD) [14].