Amniomax

A549 cells were maintained in F12 medium, and 293T, NPTr (Ferrari et al., 2003 (link)) and FLN-R cells (Friedrich-Loeffler-Institut) were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10 % FCS. Primary lung cells from the microbat (M. myotis) were grown in DMEM containing 10 % FCS, penicillin (100 units ml⁻¹) and streptomycin (100 µg ml⁻¹) and 20 % amnioMAX (Gibco).

Metaphases were obtained from fibroblast cell cultures from a male sample of S. apella and C. capucinus (Cebidae), from Catoctin Zoo, Thurmont MD, USA and the Laboratory of Genomic Diversity of the National Cancer Institute, Frederick, MD, USA. Fibroblast cells were grown for 72 h in alphaMEM culture medium (Gibco, Waltham, MA, USA), 5% Antibiotics Penicillin/Streptomicin, 15% FBS, 10% amniomax (Gibco).
Lymphoblasts of a male sample of H. sapiens were grown in RPMI culture medium, following standardised protocols to obtain metaphases.
Cells harvesting was performed after 3 h incubation of colcemid 10 μL (10 μg/mL Gibco) followed by hypotonic treatments 0.075 M KCl for 20 min at 37 °C following a protocol from Small et al. [33 (link)].

Chinese hamster ovary (CHO) cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mmol/L L‐glutamine, penicillin (50 U/mL), and streptomycin (50 μg/mL) in a humidified atmosphere at 37°C with 5% CO₂. Primary fibroblasts were obtained from punch skin biopsies. Tissue was cut into small pieces (1 × 3 mm), and cells were cultivated in a fibroblast growth medium consisting of AmnioMax (Gibco, Thermo Fisher) supplemented with 20% FBS. For long‐term culture, fibroblasts were maintained in DMEM supplemented with 10% FBS.

Mouse primary fibroblasts were isolated6 (link) and cultured in DMEM supplemented with 10% FCS or Amniomax (Gibco). Cells were pulsed with 10 μM EdU 3–4 h before fixation and staining with the Click-it EdU Imaging Kit (Invitrogen). To stimulate or inhibit specific signalling pathways in fibroblasts, the following concentrations of GFs and inhibitors were added to the medium for 24 or 48 h: Shh 1 μg ml⁻¹; TGF-β2 10 ng ml⁻¹; FGF2 20 ng ml⁻¹; FGF5 10 ng ml⁻¹ (all from RnD Systems; vehicle: PBS); IPI4182 0.5 μM (Infinity Pharmaceuticals; vehicle: DMSO); RepSox 25 μM (Tocris; vehicle: DMSO); PD173074 2 μM (Tocris; vehicle: DMSO). DED was prepared by floating 5 mm diameter punch biopsies of back skin on 0.8% trypsin (Gibco) dissolved in PBS for 60 min, separating the dermis from the epidermis with forceps and depleting cells from the tissue by at least ten freeze–thaw cycles. Before fibroblasts were seeded onto DEDs, the tissue was placed into 24-well cell culture inserts and equilibrated with medium.

Cells were split at a ratio 1:2 in a medium with 5-10 % of AmnioMax (Gibco). After two days of culturing colcemid (KaryoMAX, Gibco) was added to a final concentration of 0.1 μg/ml for overnight incubation. Three hours before cell harvesting ethidium bromide was added to a final concentration of 1.5 μg/ml. Cells were dissociated mechanically by scraping and centrifuged for 5 min at 0.6× g. Cell pellet was gently resuspended in hypotonic solution (33.5 mМ KCl, 7.75 mM sodium citrate) and incubated for 2 h at 25 °C. For prefixation treatment 1/20 volume of fresh ice-cold fixative (methanol: acetic acid - 3:1) was added, mixed carefully and incubated for 12 min at 4 °C. Then cells were centrifuged for 5 min at 0.6× g and supernatant was discarded. For cell fixation, the pellet was covered by ice-cold fixative (−20 °C) and kept for 30 min at −20 °C without mixing. Cells were then centrifuged for 5 min at 0.6× g and resuspended in ice-cold fixative. The chromosome suspensions were stored long-term at −20 °C.

Primary fibroblasts were obtained from skin biopsy from 10 individuals, 2 patients with mitochondrial MRC deficiency and 2 patients with F0F1-ATP synthase deficiency and 7 healthy controls (Table S1). Written informed consent was obtained from all participants (Ethics Committee from the Angers University Hospital approval: CPP Ouest II – Angers, France; Identification number: CPP CB 2014/02; Declaration number: DC-2011-1467 and Authorization number: AC-2012-1507). Primary human fibroblasts were cultured in 2/3 Dulbecco’s modified Eagle medium (DMEM-F12, PAN Biotech, Wimborne, UK), 1/3 Amniomax (Gibco, Invitrogen, Paisley, UK) (glucose concentration, 2 g/L and glutamine, 1 mM) supplemented with 10% fetal bovine serum (PAN Biotech, Wimborne, UK), 1 mM sodium pyruvate (Gibco, Invitrogen, Paisley, UK), and 50 μg/mL uridine (Sigma Aldrich, Lyon, France) at 37°C, 5% CO2. All experiments were conducted on fibroblast cultures between passages 6 and 25 to avoid artifacts due to senescence.²⁴ (link)