The procedure for the isolation and culture of osteoclasts described earlier by Boyde
et al. and by Chambers
et al.[
17,
18] was slightly modified and has been described in detail previously [
19]. Briefly, mechanically harvested osteoclasts from the long bones of 1 or 2-day-old Sprague-Dawley rat pups were allowed to attach to sonicated bovine cortical bone slices (0.5 cm
2). After 30 minutes, the non-attached cells were rinsed away, and the remaining cells on the bone slices were cultured in three groups: control, heptanol and Gap 27, described below. Each treatment group had five bone slices and each of the experiments was repeated three times with similar results obtained each time. The control group was cultured in DMEM (Dulbecco's modified Eagle's medium) buffered with 20 mM Hepes and containing 0.84 g of sodium bicarbonate/liter, 2 mM L-glutamine, 100 IU of penicillin/ml, 100 μg of streptomycin/ml and 7% heat-inactivated fetal calf serum (FCS), at +37°C (5% CO
2, 95% air), which medium will be later referred to as DMEM-FCS. The heptanol group was cultured in medium with 0.15% ethanol and 3 mM heptanol in DMEM-FCS. We chose not to repeat our earlier experiments with ethanol in this study, since our previous report shows that ethanol has no effect on the parameters studied here [
12]. Third group of bone cells, Gap 27 peptide group, was cultured with Gap 27 peptide at concentration 500 μM in DMEM-FCS. In all the three groups cells were cultured for 48 hours, after which the cultures were stopped by fixing the cells and bone slices with freshly made or refrigerated 3% paraformaldehyde (PFA) and 2% sucrose in phosphate-buffered saline (PBS) for 10 minutes at room temperature. Gap 27 peptide (amino acid sequence SRPTEKTIFII) was synthesised by Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, USA; purity was >95%.