Z2 coulter counter

Cells were seeded into 24-well plates (Corning) at a density of 5 × 10³ cells/cm², and cultivated for 24 h before the application of DCX/MET/FF-containing media. After 48 h, the cells were harvested and resuspended in the original culture medium. Counting was performed with a Coulter Z2 Counter (Beckman Coulter Inc., Fullerton, CA, USA). For the analyses of cell apoptosis, cells were exposed to the tested agents, harvested, resuspended in original medium, and subjected to AnnexinV/propidium iodide staining solution according to the manufacturer’s protocol (BD Biosciences [29 (link)];). Flow cytometric detection of apoptotic cells was performed with a FACSAria FACS system (Becton–Dickinson, Heidelberg, Germany). At least 5 × 10⁴ cells were analyzed for each condition.

HFL-1 human diploid fibroblasts (HDFs) were used in in vitro conditions (growth at 37 °C, 5% CO₂ and 95% humidity). The cells were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 Units/mL penicillin, 100 μg/mL streptomycin, 2 mΜ glutamine and 1% (v/v) non-essential amino acids (complete medium). The cells were constantly cultured in a culture medium supplemented with the composition (denoted as “HS” in all Figures and Tables) in two concentrations: 2 and 5 μg/mL, diluted in DMSO (dimethyl sulfoxide). The control cultures were incubated in a medium supplemented with 0.1% DMSO.
The cells were replenished with fresh media supplemented with the composition or the diluent every 72 h, and their numbers were examined using a Coulter Z2 counter (Beckman Coulter, Brea, CA, USA) until they reached senescence (approximately after 13 weeks). The media were replenished 16h before every assay. The cumulative population doublings performed by each culture were calculated using the following formula: CPD = Σ(PD) where PD = LOG(Nfinal/Ninitial)/LOG [2 (link)], where N final represents the number of cells that were measured when each culture reached confluence, and N initial represents the number of cells that were initially seeded.

Adult cardiomyocyte size was assessed with a Coulter Counter Z2 (Beckman Coulter) equipped with a 200 μm aperture and data analyzed with Accucomp software (Beckman Coulter). 20% of the isolated cells from one heart were suspended in 2 mL medium and 10 mL Beckman Isoton II diluent (Beckman Coulter) was added. The metered volume was 1 mL, main gain 32 and aperture current 0.250. From each analysis mean and median cell volumes of the cell population above the size of 20,000 fL were recorded. The cell population below 20,000 fL (equal to a spherical particle with 17 μm diameter) was contaminated with smaller cells and cell debris, and therefore discarded from the analysis (Supplementary Figure S2).

To quantify the cell number of HIOs for calculation of multiplicity of infection (MOI), an aliquot of the HIOs fragments was dissociated into single-cell suspensions using 0.05% Trypsin-EDTA (Gibco) for 5 min at 37 °C. The cells were counted using Coulter Counter Z2 (Beckman Coulter Inc., Brea, CA, USA).
For infection, HIOs were mechanically fragmented by vigorously pipetting using a P1000 to expose the apical surface to the virus inoculum as previously described [27 (link),28 (link),29 (link)]. After virus inoculation (MOI = 0.01, 0.1, or 1) with EV-A71 or mock infection, the mixture was then incubated for 2 h at 37 °C for virus adsorption. After washing, the infected HIO fragments were mixed with 20% Matrigel-organoid growth medium and seeded at 10,000 cells/well in a 96-well plate pre-coated with 20% Matrigel-organoid growth medium at 50 µL/well.
For RD cell line infection, 20,000 cells/well were cultured in 96-well plate at 37 °C for 24 h. To prepare virus inoculum (MOI = 0.01, 0.1, or 1), EV-A71 was diluted in 2% FBS-DMEM. Medium on cell monolayer was removed, and the inoculum was added for 1 h at 37 °C for virus adsorption. After washing, the monolayer was cultured in DMEM supplemented with 2% FBS.

Axenic cultures were under 12:12 light:dark cycle at 22 °C (Poliner et al., 2015). Cell counts and cell size measurements were obtained using a Coulter Counter Z2 (Beckman Coulter) using a profile with a range of 1.8–3.6 μm. N. oceanica CCMP1779 at mid‐log phase was used for RNA isolation as described previously (Poliner et al., 2015). First‐strand DNA synthesis was accomplished using SuperScript III with oligo dT(NEB). cDNAs were amplified using primers shown in Table S4 and Q5 polymerase (NEB), blunt cloned into pCR‐Blunt (Thermo Scientific) and sequenced.