Gluteraldehyde

U2OS cells were fixed with cold methanol (–20°C) for 4 min or 0.1–0.25% gluteraldehyde + 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min. Autofluorescence was quenched by 0.1% sodium borohydride (Sigma-Aldrich) after aldehyde fixation and cells permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for another 10 min. Tyrosinated α-tubulin and detyrosinated α-tubulin were immunostained using rat monoclonal anti-tyrosinated α-tubulin clone YL1/2 (1:100–500, Bio-Rad) and rabbit polyclonal anti-detyrosinated α-tubulin (1:250–2,000; Liao et al., 2019 (link)), respectively. Other primary antibodies used were human anti-centromere antibodies (ACA, 1:100–2,000, kind gift from B. Earnshaw, Welcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK, or S. Geley). GFP-tagged constructs were visualized by means of direct fluorescence. Alexa Fluor 488 (1:200–2,000, Themofisher), STAR-580, and STAR-RED (1:200, Abberior) were used as secondary antibodies, and DNA was counterstained with 1 µg/ml DAPI (Sigma-Aldrich).

Samples were fixed with 0.1 M sodium cacodylate (Sigma), 2% gluteraldehyde (Electron Microscopy Sciences), 3% PFA (Electron Microscopy Sciences), 5% sucrose buffer (Sigma) and 1% osmium tetroxide (pH 7.4) (Electron Microscopy Sciences). The samples were then dried in increasing concentrations of high-grade ethanol, followed by critical point drying using Autosamdri 815 critical point dryer and sputter coated using Cressington 208HR sputter coating with Au or Pt/Pd. Imaging was done on a Jeol 5600LV SEM, Zeiss EVO SEM or Zeiss FESEM Ultra55 microscope. For each image the total number of cancer cells, cancer cells with nanotubes, cancer cells without nanotubes, total number of nanotubes, total number of EPI–EPI membrane nanobridges, EPI–ENDO nanobridges, number of cells forming EPI–EPI nanobridges, EPI–ENDO nanobridges and number of cells positive for both EPI–EPI and EPI–ENDO nanobridges were counted. Length and width of the nanobridges were measured using the CarlZeiss TIF annotation editor. Width was measured at three different positions across the length of the nanobridges and the average width was calculated for the comparison of length and width of the nanobridges.

Subsamples (2 mL) for the enumeration of Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes (PPEs) were collected from G1 at times T₀ and T₄₈ and immediately fixed with electron microscopy grade gluteraldehyde (Electron Microscopy Sciences, Hatfield, PA) for a final gluteraldehyde concentration of 0.25% v/v. Samples were fixed in the dark for 15 min, then flash frozen in liquid nitrogen and stored at -80°C until processing. Cells were enumerated using a BD Influx cell sorter with a 488 nm Sapphire laser (Coherent, Santa Clara, CA, United States). Large particles were removed from samples prior to counting using a CellTrics^® filter with 30 μm mesh (Partec, Swedesboro, NJ, United States). Synechococcus cells were identified based on their phycoerythrin signal (orange fluorescence), and non-phycoerythrin containing cells (Prochlorococcus and PPEs) were identified using chl a fluorescence (red florescence) and forward scatter (FSC; a proxy for cell size). Data was triggered on the FSC channel, events were counted for 10 min, and data was processed using FlowJo v10.0.7 (Tree Star, Inc., Ashland, OR, United States). Net growth rates (G1) were calculated from abundances as described above.