Glass bottom plates

Phase contrast and green fluorescence images of live HeLa H2B-GFP cells, seeded on glass bottom plates (Ibidi) and incubated at 37°C and 5% CO₂, were acquired with an Eclipse Ti-E microscope (Nikon) every 4 min for 48 h. H2B-GFP allowed for the observation of morphological changes in chromatin. Mitosis length was defined as the time from chromatin condensation initiation to complete karyokinesis and cytokinesis (see exception below), the latter observed by means of phase contrast. Each cell lineage was represented as a single bar corresponding to single cells within a lineage (n = 100), according to the following criteria: Cell lineages were classified as ‘surviving’ when all the cells within the lineage survived, and classified as ‘cell death’ when at least one cell within the lineage died. When the same type of event occurred in parallel branches within a lineage, the ones scored and represented were those initiated earlier. Cell death was sub-classified as either death in interphase or death during mitosis. Polyploidization events in the surviving lineages—either by daughter cell fusion or karyokinesis without cytokinesis—were also scored. In the case of karyokinesis without cytokinesis, for mitotic length representation, mitosis completion was defined just by karyokinesis.

For mitochondrial membrane potential cells were grown for 72 h in a complete medium or EtBr-containing medium. The membrane potential was assessed by flow cytometry using TMRE (BD Pharmigen). Briefly, cells were trypsinized and resuspended at a concentration of 1 × 10⁶ cells/ml in media containing 100 nM TMRE or 100 nM TMRE + 60 µM CCCP as control and incubated 30 min at 37 °C. Cells were washed twice in PBS containing 2% FBS and 2 mM EDTA, filtered using a 30 µm cell strainer, and measured using a BD FACS Canto II Flow Cytometer (BD Biosciences). At least 20,000 events from each sample were recorded. Data were analyzed using FlowJo 10.8.1 software (BD Biosciences). For the analysis of membrane potential in Airy Scan, cells were grown in glass bottom plates (Ibidi) and incubated for 1 h in medium containing 15 nM TMRE. Images were acquired with Airy Scan Confocal Microscope (Zeiss) with ×63 Plan-Apochromat/1.4 Oil DIC. For cristae architecture, cells were treated as described before and stained for 30 min with 250 nM PK Mito Orange dye (GenVivo Biotech) and imaged after 2 hours with TCP SP8 gSTED Leica Microsystems using PL Apo ×100/1.40 Oil STED Orange.

BMDCs were plated in glass bottom plates (Ibidi) and synchronised, as previously described. At 12 h and 24 h post synchronisation, BMDCs were washed with PBS and labelled with either Fluo-4 AM (8 µM) (Invitrogen) rhodamine-2 AM (4 µM) (Invitrogen), which are cytosolic and mitochondrial calcium indicators respectively, for 1 h at 37 °C in 5% CO₂. Following incubation with calcium stains, cells were rinsed six to seven times with cold PBS, replenished with fresh DMEM and incubated for 20 min before imaging. Ca²⁺ imaging was conducted at room temperature on a Leica SP8 scanning confocal microscope (Wetzlar, Germany), using a 20× objective. Cell images were analysed with the ImageJ software.