Lsfortessa x 20 flow cytometer

The cell cycle assay was based on a univariate analysis of DNA content upon staining with propidium iodide [29 (link)]. Cells were seeded at a density of 3.0x10⁵ cells/ml in 25cm² culture dish with or without fractions for 24 hours and centrifuged at 1700 rpm for 10 mins. Cell pellets were suspended in 1.5 ml of 1x phosphate-buffered saline (PBS) and vortexed well. A 3.5 ml of absolute ethanol was added (final concentration of 70%) to fix cells at -20°C for 1 hour. Cells were centrifuged at 1700 rpm for 10 mins. Cell pellets were suspended with 200 μl of guava cell cycle reagent that contained propidium iodide (EMD, Millipore). Suspended cells were added to wells containing the same volume of fresh guava cell cycle reagent. Cells were incubated for 30 mins in darkness at room temperature. Distribution of cells at distinct cell cycle phases was measured with the BD LSFortessa X-20 flow cytometer.

Trypanosome cells were seeded at a density of 3.0 × 10⁵ cells/ml in a 25 cm² culture dish with fractions for 24 hours and centrifuged at 1700 rpm for 10 mins. Cell pellets were suspended in 1.5 ml of 1x phosphate-buffered saline (PBS) and vortexed well. A 3.5 ml of absolute ethanol was added (final concentration of 70%) to fix cells at −20°C for 1 hour. Cells were centrifuged at 1700 rpm for 10 mins. Cell pellets were suspended with 200 μl of guava cell cycle reagent containing propidium iodide (EMD, Millipore). The distribution of cells at distinct cell cycle phases was measured with the BD LSFortessa X-20 flow cytometer.