Distilled h2o

At the end of culture with serum, C-28/I2 and T/C-28a2 chondrocytes were changed to serum-free medium for 2 hours, cells were incubated alone or with a range of concentrations (10⁻¹¹ M, 10⁻⁹ M and 10⁻⁷ M) of letrozole (Femara; Novartis Oncology, Nuremberg, Germany) during the two serum-free days (days 6 and 7 or days 8 and 9, respectively). For this purpose, stock solutions of 10⁻¹ M letrozole (EtOH-soluble) were prepared and dissolved stepwise with distilled H₂O (EMD Millipore, Billerica, MA, USA). All experiments were performed in triplicate, and controls were incubated with water instead of the aromatase inhibitor letrozole.

SON neurones were acutely dissociated by enzymatic and mechanical treatments as described previously [8] (link), [11] (link) with some modifications [14] (link), [27] (link). In brief, SON tissues (1 mm long, 0.5 mm thick, 0.5 mm wide) were dissected and enzymatically dissociated by incubation for 30 min in oxygenated HEPES-buffered normal Locke’s solution (NL; in mM: 140 NaCl, 5 KCl, 2CaCl₂, 1 MgCl₂, 10 glucose, 10HEPES, pH was adjusted to 7.25 with Tris; the osmolarity was 298–300 mOsm/l; temperature 37° C) supplemented with 1 mg/ml deoxyribonuclease I, 0.5 mg/ml proteases X, and 0.5 mg/ml protease XIV. Unless stated otherwise, most of the chemicals were purchased from Sigma-Aldrich (St. Louis, USA). After incubation, tissues were washed with NL and triturated gently using a Gilson-Pipetman (1 ml) with polypropylene white pipette-tip to isolate SON cells. Cells were plated onto glass bottom dishes (22 mm in diameter, 0.17 mm in thick: WillCo Wells-Amsterdam). Unless otherwise stated, all stock solutions of the drugs used in this study were dissolved in this total ion-free distilled H₂O (EMD Millipore Corporation, Germany). The hypotonic solution was prepared by reducing the appropriate Na⁺ concentration and in the hypertonic solution the osmolarity was increased by adding mannitol.