Facscantotm cytometer

Cell cycle was analyzed by flow cytometry by quantitation of DNA content after propidium iodide (PI) dye staining. Briefly, after seeding and overnight recovering, sensitive (MES-SA) and resistant (MES-SA/Dx5) cells were exposed for 72 h to PC and TR extracts at the concentrations of 12.5, 25, and 50 µg/mL. At the end of the treatments, cells were harvested and washed 3 times with ice-cold PBS before fixation/permeabilization with 70% ethanol at −20 °C. After 24 h, cells were washed with PBS and then stained for 1 h at 37 °C with a solution containing 200 μg/mL RNase A and 50 μg/mL PI (Sigma-Aldrich, St. Quentin Fallavier, France). At the end of the incubation, cells were washed with cold PBS, and then PI fluorescence, varying according to DNA content and cell cycle phase, was measured with a BD FACSCanto^TM cytometer equipped with BD FACSDiva software v.8.0.3 (BD Biosciences, Le Pont de Claix, France) and analyzed with the v.10 FlowJo software (Tree Star, Ashland, OR, USA).

Cell cycle analysis was performed by staining single live cells with propidium iodide (Sigma, cat. P4170), according to the manufacturer's instructions. Samples were analyzed by flow cytometry using a BD FACSCanto^TM cytometer.

For tracking of labeled EVs and phenotypical analysis of murine organs, femurs were flushed and single-cell suspensions were filtered through a 40-μm strainer. Cells were washed in PBS with 1% BSA and incubated with anti-CD11b-PerCP-Cyanine5.5 (clone M1/70, 1:100, BD Biosciences – 561114) at predetermined saturating concentrations. PKH67-labeled EV-positive cells were detected using blue laser excitation and 488 nm emission. Data for 1,000,000 cells was acquired on a BD FACS Canto^TM cytometer with Diva software (BD) and was analyzed using FlowJo^TM software (TreeStar).