Nucleocounter nc 3000 system

Cell cycle analysis was performed based on the DNA content of the cells. Briefly, the cells were treated with selumetinib (100 nM), BEZ235 (10 nM), or both for 72 h in 6-well plates. Then, both supernatant and attached cells were collected and mixed with lysis buffer + DAPI for 5 min at 37 °C. After the application of stabilization buffer, the samples were analyzed by NucleoCounter NC-3000 system (Chemometec, Allerod, Denmark) following the manufacturer’s protocol. In simultaneous parallel experiments, the cell number was determined at the end of the treatment by the NucleoCounter NC-3000 system (Chemometec). After trypsinization, the cells were stained with Acridine Orange and DAPI (Solution 13, Chemometec, 910–3013). Then, 10 µL of each sample was loaded in an 8-well NC-slide and the cells were counted.

Vitality assay was estimated using NucleoCounter^® NC-3000™ system (ChemoMetec A/S) based on an assessment of the reduced thiol group concentrations, such as reduced glutathione (GSH). Briefly, 4 × 10⁹ cells/mL, control or treated with HA extracts (0.1, and 1 µg/mL) for 24 h, were mixed with VitaBright-48 iodide (ChemoMetec A/S), acridine orange iodide (ChemoMetec A/S), and propidium iodide (ChemoMetec A/S) and immediately analyzed with the NucleoCounter^® NC-3000™ system according to the instructions provided by the manufacturer.

Mitochondrial transmembrane potential (Δψm) was measured using the cationic dye JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide) by advanced image cytometer NucleoCounter^® NC-3000™ system (ChemoMetec, Copenhagen, Denmark). This method is based on fluorescent detection of healthy and apoptotic cells. In apoptotic cells, Δψm collapses and JC-1 localizes to the cytosol in its monomeric fluorescent form. Briefly, 1.5 × 10⁶ cells/mL, control or treated with HA extracts (0.1 and 1 µg/mL) for 24 h, were mixed with 200 μg/mL JC-1 (ChemoMetec A/S) and incubated in 37 °C for 10 min. After washing procedures, samples were resuspended in 1 μg/mL of 4′,6-diamidino-2-phenylindole (ChemoMetec A/S) and analyzed with the NucleoCounter^® NC-3000™ system.

Vitality assay describes changes in the intracellular level of (reduced) thiols such as glutathione which exists in two forms: reduced – GSH and – oxidized state (GSSG). The decrease in GSH levels is an early signal of cell death caused either by the direct GSH oxidation promoted by radicals or by the export of GSH through an ATP-dependent plasma membrane transport system. The assay was performed using NucleoCounter^® NC-3000^TM system (ChemoMetec, Denmark) according to manufacturer’s protocol. Cells incubated with studied concentrations of polyphenols were detached from the plates, washed and stained with Solution 5 (containing three various reagents: VitaBright-48, propidium iodide, and acridine orange). Cisplatin (10 μM) was used as a positive control. Thereafter, the intensity of fluorescence was measured using NucleoCounter^® NC-3000^TM.

CYTO-ID® Autophagy Detection Kit (ENZ-51031, Enzo) was used to measure autophagic activity according to the manufacturer’s instructions. 2.4×10⁵ cells were seeded into each well of six-well plates and grown in a CO₂ incubator at 37°C overnight. The next day, MG or vehicle was applied to the cells for 24 h. Number of autophagic vacuoles was measured and the autophgic flux was monitored after the cells were harvested and stained with fluorescent dyes. The fluorescence intensity and number of autophagosomes were detected and measured using the NucleoCounter^® NC-3000^TM system (ChemoMetec) [37 (link)].

MRC-5 and A-549 cells were incubated with 0.4–0.7 μM auranofin for 24, 48, and 72 h (at a density of 5 × 10⁵ cells/mL). Cells were trypsinized for cell cycle analysis and then washed twice with cold PBS. Cells were centrifuged for 5 min at 1500 rpm. Next, cells were resuspended with 580 μL PBS solution containing 10 μg/mL of DAPI (solution 10, Chemometec), incubated for 5 min, and analyzed by flow cytometry. Cell cycle analysis was performed using Nucleo Counter^® NC-3000^TM system (Chemometec).
Cells (2 × 10⁵ cells/well) were seeded in 24-well plates to grow in a monolayer for 24 h. Then, a sterile 2–20 μL pipette tip was held vertically to scratch a cross in each well. The detached cells were removed by washing with 500 μL PBS and shaking at 500 rpm for 5 min. 500 μL of fresh medium with auranofin (in the range of 0.4–0.8 µM to achieve a concentration equal to IC50 and in the range of 0.8–1.6 µM to achieve a concentration equal to 2 × IC50) was added to each well and incubated for 72 h. Before the image acquisition, the plate was washed with 500 μL pre-warmed PBS [62 (link)] and gently shaken for 30 s. Then, a medium was added again, and pictures were taken. The scratch closure was monitored and imaged in 24 h intervals using an SZX10 microscope (Olympus, Tokyo, Japan). Images were analyzed using ImageJ software with Wound Healing Tool.

Lysosome formation induced by MG was measured using the LYSO-ID® Green Detection Kit (ENZ-51034, Enzo). 2.4×10⁵ cells were seeded into each well of six-well plates and cultured in a CO₂ incubator at 37°C overnight. MG or the vehicle were used to treat the cells for 24 h, and the cells were harvested and stained with fluorescent dyes using the LYSO-ID® Green Detection Kit as described by the manufacturer’s. Fluorescence intensity was measured using the NucleoCounter® NC-3000^TM system (ChemoMetec) [37 (link)].