Cyflow cube 6 flow cytometer

To determine the impacts of extracts on HGEC proliferation, HGEC were cultured in a 96-well plate after overnight incubation at the density of 8 × 10³ cells/100 μL/well. After exposure to the extracts for 24 h, cellular morphology was recorded by an inverted microscope (TS-100, Nikon, Japan). Simultaneously, a cell counting kit-8 assay kit (CCK-8, Yi Fei Xue Biotechnology, China) was employed to analyse cell proliferation. To calculate cell viability, the absorbance was measured at 450 nm with a microplate reader (Molecular Devices LLC, USA).
Annexin V-FITC/PI apoptosis assay kit was used for cell apoptosis analysis. In brief, HGEC were seeded in 6-well plates (1 × 10⁶ cells per well) with DMEM for 24 h. After that, the spent DMEM was replaced with the exposure solution and treated for 24 h. After the exposures, cells were harvested and washed with cold phosphate buffer saline (PBS) three times and then immersed in 500 μL of PBS binding buffer (containing 5 μL Annexin V-FITC and 5 μL of PI-staining solution) for 15 min at room temperature in the dark. Approximately 1 × 10⁴ cells were loaded with a CyFlow^® Cube 6 Flow cytometer (Sysmex Partec, Germany) to assess cell apoptosis, and then analyzed by Flow Jo Version 10.0.6 software (BD Biosciences, USA).

The efficiency of the electroporation protocols was by assessing the amount of the Yo-Pro^TM-1 Iodide fluorescent dye absorbed by the cell. Initially, cells were trypsinized and suspended in calcium-depleted electroporation buffer—SMEM (5 × 10⁴ cells/400 µL), and Yo-Pro™-1 Iodide (YP-1, λ_exc491/λ_em509, Thermo Scientific^TM, Warszawa, Poland) was added. Cell suspensions were then transferred to cuvettes and electroporated. Then cells were washed by DPBS to remove the content of the free dye, gently centrifuged 2 min × 100× g, and suspended in 500 µL of DPBS for measurements in polystyrene FACS tubes, 10⁴ events/sample were measured in triplicate. Flow cytometric analysis was performed by using the CyFlow CUBE-6 flow cytometer (Sysmex, Warszawa, Poland). Data was analyzed using CyView Sofware (Sysmex, Warszawa, Polska).

Flow cytometry analysis was performed to evaluate electroporation efficacy through the assessment of the ability of cells to internalize impermeant dye – Yo-pro-1. Immediately before EP, YO-PRO™-1 iodide (YP-1, λ_exc491/λ_em509, Thermo Scientific, Poland) was added to the cell suspension. Concentration of YP-1 in the STM or HEPES buffer was 1 μM. Flow cytometric analysis was performed using CyFlow CUBE-6 flow cytometer (Sysmex, Poland). The samples were excited using the 488-nm line of the blue laser and the fluorescence of YP-1 was measured with FL-1 detector. Data were analyzed using CyView software (Sysmex).