Dulbecco s phosphate buffered solution dpbs

A compressive force of 2 g/cm² was applied according to the protocol of Kirschneck et al. [41 (link)] and as previously reported [42 (link)]. Briefly, in tandem with LPS applications, glass plates were placed on fatty acid-cultured HPdLFs for 6 h at 37 °C, under 5% CO₂ and 95% humidity. Afterwards, the cells were either directly processed using TRIzol^TM Reagent (Thermo Fisher Scientific) for expression analysis or isolated using Dulbecco’s Phosphate-Buffered Solution (DPBS, Thermo Fisher Scientific) for histone extraction and, subsequently, protein analysis. In 48-well plates, the compressive force was applied via six-hour centrifugation at 30 °C with a force of 7.13 g/cm². Control cells were cultured at 30 °C for the duration of force application.

Animal experiments were approved by the Institutional Animal Ethics Committee, Indian Institute of Science, Bangalore, India. All animal protocols were performed in accordance with the guidelines for care and use of laboratory animals set by Indian National Science Academy. Six to eight -week-old CD-1 female mice, weighing 25–28 g, were used for the isolation of BM-MSCs BM-MSCs were isolated as previously described (PMID: 29760732). Briefly, the mice were sacrificed by cervical dislocation and the femurs and tibias were dissected out. The bone marrow was flushed with Dulbecco's phosphate-buffered solution (DPBS; Thermofisher scientific, Waltham, USA). The cell suspension was filtered through a 70 μm cell strainer (BD Falcon, USA), and centrifuged at 300×g for 10 min. The cell pellet was suspended in 1 ml MSC culture medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM glutamine (all purchased from Thermo Scientific, Waltham, USA). Cells were seeded in 35 mm cell culture dishes at a density of 1 × 106 cells/cm2 and incubated in a humidified incubator at 37 °C, 5% CO₂. After 1 day, nonadherent cells were removed by washing twice with DPBS and fresh MSC culture medium was added. The medium was replaced with fresh MSC culture medium every 2 days.

C57Bl/6 mice (n = 82, 4 weeks old) were obtained from Charles River Laboratories (USA). The ovaries were removed through small dorsolateral skin incisions and were placed either in Leibovitz L-15 medium (Lonza, Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) (transport solution for slow freezing (SF)) or in Dulbecco’s phosphate-buffered solution (DPBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) supplemented with 20% FBS (transport solution for vitrification (VT)). Leibovitz L-15 is the conventional medium for the SF procedure, whereas there is no consensus for ovarian tissue VT. Therefore, we used Youm’s VT procedure^{30 (link)}. In order to cryopreserve only the ovary, adjacent tissues taken at the time of ovariectomy were removed under a binocular using a scalpel. The Animal Ethics Committee of the University of Liège approved this study (# 1547) and all experiments were performed in accordance with relevant guidelines and regulations.