Attune nxt analysis software

HeLa, OV2008, Caov-3, and SK-OV-3 cell lines were seeded in 60 mm dishes at a density of 300,000 cells/well and exposed to trabectedin (IC₅₀) and/or propranolol (HeLa - Caov-3: 50 μM, OV2008 - SK-OV-3: 100 µM) alone or in combination in the absence or presence of 10 µM of NE. Afterward, the cells were harvested, washed twice in cold PBS, fixed in 70% ethanol, and stored at −20°C until analysis. The cell cycle modulation by drugs was evaluated by propidium iodide (PI) staining, as previously described (Danza et al., 2021 (link)). Then, flow cytometry (FCM) analysis was performed by using the Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, MA, United States), and cell cycle phases were analyzed by the Attune NxT Analysis Software (Thermo Fisher Scientific, MA, United States).

The human pancreatic cancer cells were seeded in 60 mm dishes at a density of 300,000/well and incubated at pH 6.8 and for 24 h, or 24 h followed by 48 h, washing out with Gem@TpHResMic or free Gem. Gem was used at 3 µM, 5 µM, and 54 nM in PANC−1, MIAPaCa-2, and AsPC−1 cells, respectively. Then, the cells were harvested, washed twice in ice-cold PBS pH 7.4, fixed in 4 mL of 70% ethanol, and stored at −20 °C until analysis. The cell cycle modulation induced by treatments was studied, as previously described [42 (link)], by propidium iodide (PI) staining; the pellet was resuspended in PBS without Ca²⁺ and Mg²⁺, containing 1 mg/mL RNase, 0.01% NP40, and 20 µg/mL PI (Sigma); then, FCM analysis was performed by Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Data were interpreted using the Attune NxT Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA).

PD1 and PD-L1 expression analysis was performed by FCM in OC and CC cell lines at the basal level and after 24 h treatment with trabectedin and propranolol alone and in combination under stress stimuli by 10 µM of NE. Cells were seeded in 60 mm dishes at a density of 500,000/well and treated after 24 h. Afterward, the cells were harvested, washed twice, resuspended in ice-cold PBS without Ca²⁺ and Mg²⁺, and incubated to labeling with anti-human SuperBright 702-PD1 and anti-human PE-Cyanine 5-PD-L1 antibodies for 30 min at 2°C–8°C in the dark. After staining, the cells were washed with PBS without Ca²⁺ and Mg²⁺ and analyzed by using the Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, United States) equipped with four lasers (405 nm (violet), 488 nm (blue), 561 nm (yellow), and 637 nm (red)) for sample reading. The data were analyzed using the Attune NxT Analysis Software (Thermo Fisher Scientific, Waltham, MA, United States).