Cytoflex system b4 r2 v0

Flow cytometry samples were analyzed in flow cytometer Cytoflex System B4-R2-V0 (Beckman Coulter), equipped with 488 nm solid state laser (50 mW), FS, SS, FITC (525/40 BP), PE (585/42), ECD (610/20 BP), and PC5.5 (690/50 BP) channels. Twenty thousand cells were analyzed per sample at low flow rate. Flow cytometry analyses was performed with FlowJo^® 7.6 and CytExpert Data software.
The microscopy samples were analyzed on an Olympus BX6F2 microscope with appropriate filter cubes: U-FDICT (differential interference contrast), U-FYW (far-red), U-FGNA (red), and U-FUNA (blue), with a 60× oil immersion objective. Images were processed using Olympus ImageLS software.

Cells were incubated with an IC₅₀ concentration of compounds and harvested 24 and 48 h later. Cells were then fixed with ice-cold ethanol (70%) for 30′ on ice, washed with PBS, and the supernatant was discarded after centrifugation. The samples were then treated with ribonuclease A (200 mg/mL) for 15′ at 37 °C and stained with PI (50 µg/mL) for 30′ in the dark at room temperature before the analysis [32 (link)]. Stained cells (20.000 single-cell events) were analyzed and sorted based on the area and pulse width of the PI signal in a flow cytometer, Beckman Coulter (Cytoflex System B4-R2-V0). Cell-cycle analyses of DNA histograms were evaluated using CytoExpert Software version 2.4 (Indianapolis, IN, USA).