Fv1000 live

The intracellular molecular uptake efficiency was measured by a cell cytometer (MoFlo XDP, Beckman Coulter, USA). The cell was regarded as “fluorescing” if its fluorescence intensity was greater than the background signal from 99% of the untreated control cells. The fluorescence intensity of a bulk sample was measured by a spectrofluorophotometer (RF-5301 pc, Shimadzu, Japan) with an excitation wavelength of 488 nm when detecting calcein. The image for cellular uptake was taken by confocal laser scanning microscopy (FV1000 Live, Olympus, Japan) with an excitation wavelength of 488 nm.
Cell viability after electroporation was assayed by measuring the quantum yield (QY) of chlorophyll in photosynthetic unicellular eukaryotes. QY was measured by AquaPen-C APA100 (Czech Republic). The electroporated samples were incubated for one hour in the dark for dark adaptation. Normalized viability was calculated by dividing the QY of the experimental group by the QY of control group.

The hIOs were treated for 1 h with 5 μM Fluo-4 acetoxymethylester (Fluo-4 AM; Molecular Probes, Eugene, Oregon, USA) and washed with Ca²⁺-free isotonic buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl₂, 5.5 mM D-Glucose). The hIOs were mounted on a confocal microscope (FV1000 Live; Olympus) and stimulated with 50 mM glucose (Sigma-Aldrich) in Ca²⁺-free isotonic buffer. hIOs were excited at 488 nm, and the signal emitted at 505 to 530 nm was recorded. The fluorescence intensity of the region of interest (ROI) was calculated using FV1000 software.

Localization of pVEC and Cas9 was observed using confocal laser scanning microscopy (FV1000 Live, Olympus, Japan). Peptron Inc. also prepared the fluorescein-5-isothiocyanate (FITC)-conjugated pVEC. Cy3-conjugated Cas9 was purchased from ToolGen, Inc (Korea). As mentioned above, the trypsin was treated before the microscopic analysis because pVEC induces strong interaction between foreign protein and the cell surface even though foreign protein is not actually delivered into the cell^{23 (link)}. The delivery efficiency was measured by a cell cytometer (FACS Calibur, BD Biosciences, USA), and the cell was regarded as “fluorescing” when its fluorescence intensity was higher than the background signal from 99% of the untreated control cells. The quantum yield for estimating the cell viability was measured using an AquaPen-C APA100 (Photon System Instruments, Czech Republic). Before measuring the quantum yield, the sample was incubated in the dark for one hour as a dark adaptation.