Accuri c6 cflow software

The intact and TR-57-treated SUM159 cells were washed three times with PBS and detached from the surface of the culture plastic using a 0.05% trypsin-EDTA solution. Cell aliquots containing equal cell amount (500,000 cells) were stained for 30 min in a CO₂ incubator in the full incubation medium (DMEM/F12) with 10 nM 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) to measure the mitochondrial membrane potential or with 50 nM MitoTracker Deep Red to assess mitochondrial mass. Experimental data were obtained using a BD Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA) were processed using BD Accuri C6 CFlow software 1.0.264.21 (BD Bioscience, Franklin Lakes, NJ, USA) and resulting histograms were plotted using FlowJo v10 software (BD Bioscience, Franklin Lakes, NJ, USA).

Subsamples for flow cytometry were taken directly from the Niskin bottles, fixed with glutaraldehyde (1.0% final concentration), incubated for 15 min at 4°C and finally stored at −80°C until further analysis. Bacterial cell numbers were determined using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) by SybrGreen I (SGI, Invitrogen, Carlsbad, CA, USA) staining. 1 μm Multicolour latex beads (Polysciences Europe, Eppelheim, Germany) were used as an internal standard. After 30 min of incubation in the dark, each sample was analysed using a flow rate of 14 μl min⁻¹. Bacteria were detected using manual gating after visual inspection of the dot plots of the green versus red fluorescence and forward versus sideward scatters (FSC and SSC) and their histogram plots, respectively. The internal fluidics calibration of the device was used for volume calibration and verified by TruCount beads (BD Biosciences, Franklin Lakes, NJ, USA) as described previously [42 (link)]. Data were processed by BD Accuri C6 C-Flow software (version 1.026421).