Yo pro 1 iodide yp

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).

On the day of experiments, cells were embedded in an agarose gel three-dimensional (3D) culture, similar to previously described methods^{57 (link)}. The bottom of a 60 mm dish was coated with 7 mL of 2% low-gelling-temperature agarose (Sigma-Aldrich, St. Louis, MO) in F-12K complete medium. Cells were harvested and resuspended in 0.75% agarose in the F-12K complete medium at 5 × 10⁶ cells/mL; 4 mL of this suspension was deposited dropwise over the 2% agarose base layer in a 60 mm dish. The dishes were incubated at 4 °C for 5 minutes to hasten agarose jellification and prevent cell sedimentation, and then transferred to the incubator for at least 30 minutes before nsEP exposure. YO-PRO-1 iodide (YP; 1 µM in 3 mL PBS; Thermo Fisher Scientific, Waltham, MA) was added to each dish 5 minutes prior to nsEP exposures and incubated at 37 °C to allow the dye to equilibrate throughout the agarose gel.