Te300 inverted fluorescent microscope

The SKBR3, MCF7, and 4T1 cells were seeded at 15,000, 15,000, 8000 cells per well in 48-well plates, respectively, for 16 h. The NP-Chi-xPEI-DNA complexes prepared at a 10:1 wt/wt ratio of [Fe] NP-Chi-xPEI:DNA were added to 200 μL of fully supplemented culture media to give a final DNA concentration of 2 μg/mL in each well. The cells were incubated with complexes for 48 h and the cell culture media were replenished after 24 h. Transfections using the commercial agent, Lipofectamine 3000, was performed following the manufacturer’s protocol. A DNA concentration of 2 μg/mL was used for all the transfection agents for the transfection study. The cells were imaged 72 h post-transfection with a Nikon TE300 inverted fluorescent microscope (Nikon, Tokyo, Japan).

The SKBR3, MCF7, and 4T1 cells were seeded at 15,000, 15,000, 8000 cells per well in 24-well plates, respectively, and incubated for 16 h. For the cellular uptake study, NP-Chi-xPEI was complexed with Cy5-labeled DNA at 10:1 wt/wt [Fe] NP-Chi-xPEI:DNA ratio before adding to cells at 1 μg/mL DNA concentration. For the DNA release study, NP-Chi-xPEI and DNA were labeled with NHS-AF488 and Cy5, respectively, before forming a complex at 10:1 wt/wt [Fe] NP-Chi-xPEI:DNA ratio. The NHS-AF488 and Cy5-dually labeled NP-Chi-xPEI-DNA complexes were added to cells at 1 μg/mL of DNA concentration. Fluorescently labeled NP-Chi-xPEI-DNA complexes were incubated with cells for 24 h before imaging with a Nikon TE300 inverted fluorescent microscope (Nikon, Tokyo, Japan) in both studies.

Cells were fixed with 3.7% (v/v) paraformaldehyde (Electron Microscopy Services, Hatfield, PA) in PBS for 10 minutes and washed 2 × 3 minutes with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS, followed by a PBS wash. Following blocking in 1% BSA PBS, cells were incubated with AlexaFluor phalloidin 568 (Thermo Fisher Scientific, no. A12380) (5 units/200 μl in 1% BSA PBS per well) for 20 minutes. Cells were washed with PBS three times, wherein the first wash contained DAPI. Cells were mounted in Slow Fade Gold Anti-Fade reagent (Thermo Fisher Scientific) and imaged with a Nikon TE300 inverted fluorescent microscope (Belmont, CA).