Novocytetm 2000 flowcytometry system

Viability (live/dead) assessment was performed by staining cells with 7-aminoactinomycin D (7-AAD) (Biolegend). After treatment, cells were harvested, washed 1 to 2 times with phosphate-buffered saline (PBS), and centrifuged at 400× g for 5 min. Cell pellets were resuspended in 200 µL of flow cytometry staining buffer (PBS without Ca²⁺ and Mg²⁺, 2,5% FBS) and stained with 5 µL of 7-AAD staining solution for 15 min at room temperature in the dark. Samples were acquired on the NovoCyteTM 2000 FlowCytometry System (ACEA) equipped with a 488 nm argon laser. Detection of 7-AAD emission was collected through a 675/30 nm filter in the FL4 channel.

Cytotoxicity tests were carried out following a known procedure [40 (link)]. Viability (live/dead) assessment was performed by staining cells with 7-AAD (7-aminoactinomycin D) (Biolegend, San Diego, CA, USA). The cells were seeded in a 24-well plate (1 × 10⁵ cells/well). After treatment (24 h), the cells were harvested, washed 1–2 times with phosphate-buffered saline (PBS), and centrifuged at 400 g for 5 min. Cell pellets were resuspended in 200 mL of a flow cytometry staining buffer (PBS without Ca²⁺ and Mg²⁺, 2.5% FBS) and stained with 5 μL of a 7-AAD staining solution for 15 min at room temperature in the dark. Samples were acquired on a NovoCyteTM 2000 FlowCytometry System (ACEA, San Diego, CA, USA) equipped with a 488 nm argon laser. Detection of 7-AAD emission was done using a 675/30 nm filter in the FL4 channel.

Cell cycle was analyzed using the method of propidium iodide staining. Briefly, cells were plated in 24-well round bottom plates at density 10 × 105 cells per well, centrifuged at 450× g for 5 min, and fixed with ice-cold 70% ethanol for 24 h at 0 °C. Cells were then washed with PBS and incubated with 250 μL of Guava Cell Cycle Reagent (Millipore, Burlington, MA, USA) for 30 min at room temperature in the dark. Samples were analyzed on NovoCyte^TM 2000 FlowCytometry System (ACEA, San Diego, CA, USA).