Nucleocounter nc 3000 image cytometer

Cell apoptosis assay was performed using NC 3000 system (ChemoMetec USA Inc., Lillerød, Denmark) according to the manufacturer’s protocol for Annexin V Assay. The A375 cells were seeded in 6-well culture plates at a density 1 × 10⁵ cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC₅₀ values or DMSO as vehicle in control cultures and analysed after 24h incubation. The cells were dissociated into single-cell suspensions in PBS (Corning, New York, NY, USA) and app. 4 × 10⁵ cells were resuspended in 100µl of Annexin V binding buffer and incubated with Annexin V—CF488A conjugate and Hoechst 33,342 for 15 min at 37 °C. Next, the cells were washed with Annexin V binding buffer and the cell pellets were resuspended in 100 mL of Annexin V binding buffer supplemented with 10 µg/mL of propidium iodide (PI) and Annexin V Assay was performed immediately. Cellular fluorescence was quantified using a NucleoCounter^® NC-3000™ image cytometer (ChemoMetec USA Inc., Lillerød, Denmark). Data were derived from three independent experiments with three replicates for each concentration of tested compounds.

To study the apoptotic effect of cordycepin, we analyzed the PI-Annexin-V staining pattern by using the Annexin-V-FLUOS staining kit (Roche Diagnostics). Briefly, cells were treated with cordycepin (20, 40, 60, and 80 μg/ml) for 48 h, collected by scraping, and then washed twice with PBS. The cell suspension was centrifuged at 2,000 rpm for 2 min and incubated with 0.2 mg/ml Annexin-V FLUOS and 1.4 mg/ml PI for 15 min at room temperature. Then, the cells were analyzed on a NucleoCounter^® NC-3000^™ image cytometer (ChemoMetec, Copenhagen, Denmark) using an excitation wavelength of 488 nm, with a 530/30 nm band-pass filter and a 670 nm high-pass filter for detecting Annexin-V and PI, respectively.

Briefly, the human lung epithelial adenocarcinoma line A549 was cultured in 96-well plates (1 × 10⁵ cells/well) with S. pneumoniae bacteria or EMVs for 24 h, after which cell viability and apoptosis were measured as described previously [7 (link)]. The culture medium was RPMI 1640 (Gibco, Waltham, MA, USA) supplemented with heat-inactivated 10% FBS and antibiotics (Gibco). The ranges of multiplicity of infection (MOI) of S. pneumoniae that were used to infect the A549 cells were 0.001, 0.1, 10, and 1,000 for S. pneumoniae BAA-255 and 0.001, 0.01, 0.1, and 1 for S. pneumoniae KCCM-41569. The concentrations of S. pneumoniae BAA-255 EMVs were 50, 100, and 200 μg protein in 100 μL of culture medium. To measure cell viability, the cells were stained with acridine orange and DAPI (ChemoMetec, Allerød, Denmark). To measure apoptosis, the cells were stained with FITC-conjugated Annexin V, propidium iodide, and Hoechst (ChemoMetec) according to the manufacturer's instructions. The stained cells were then analyzed in a NucleoCounter NC-3000 image cytometer (ChemoMetec).