Imagestreamx mk 2 flow cytometer

The assessment of cell cycle analysis involved the following steps: trypsin dissociation of MG-63 cells, subsequent rinsing with an assay buffer to maintain solution equilibrium, and suspension of cell pellets in a new assay solution at a concentration of 1 × 10⁶ cells/mL. Subsequently, in preparation for propidium iodide labeling, cellular permeabilization was achieved by treating the cells with 1000 μL of a cell fixation agent, thus halting any ongoing metabolic activities. The fixing agent was eliminated using a centrifugation process lasting 5 min at a force of 500× g. Subsequently, the cells were immersed in a propidium iodide staining solution and afterwards subjected to incubation in a light-deprived environment at room temperature for a duration of 30 min. The MG-63 cell cycle was analyzed using the ImageStreamX Mk II flow cytometer (Merck KGaA, Darmstadt, Germany).

A FacsAria II Sorp flow cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a 70 μm nozzle was used for the estimation of the fraction of species-specific cell numbers and the calculation of cellular chl a concentration in the bi-algal cultures (Dunker et al., 2013 (link)). The Chl a fluorescence signals were used for the differentiation of the strains during co-cultivation. Chl a fluorescence was excited at 488 and 532 nm and the respective fluorescence emission was measured at 670 nm (LP) and 670 nm (BP 14 nm) which allowed to distinguish M. aeruginosa from green algae in two separate clusters due to their different pigmentation pattern (Dunker et al., 2013 (link)).
In general at least 5000 events were collected for data analysis, with only a limited number of exceptions. Flow cytometric data were analyzed with the FlowCore-package and visualized by FlowViz-package of R-software. Prior to analysis data were bi-exponentially transformed.
The accumulation of enzymatic resistant empty cell walls was measured with an ImageStream^®X Mk II flow cytometer (Merck Millipore, Darmstadt, Germany), which allows to identify single events of a cytogram by high throughput image analysis. Chl a fluorescence (Ex. 488 nm/Em. 702/85 nm) and cell area (μm²) were used to distinguish between empty cell walls and vital cells.