Midi prepped endotoxin free plasmid dna

A glass-bottom 24-well plate (MatTek Corporation , Ashland, MA) was coated with 1X poly-D-lysine (Sigma Aldrich), rinsed with water, and allowed to dry at room temperature. HEK-293A or ARPE-19 cells were plated at a density of 100,000 cells/well and transfected the following day with 500 ng of midi-prepped endotoxin-free plasmid DNA (Qiagen). Forty-eight hours after transfection, fresh media was added, and 24 h later (72 h post transfection), the cells were washed twice with 1X PBS (Fisher BioReagents, cat# BP2944100, Waltham, MA) followed by incubation with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) for 20 min. After PFA incubation, cells were washed with 1X PBS. For the ARPE-19 cells, the cell nuclei were stained with 300 nM 4’,6-diamidino-2-phenylindole (DAPI), dilactate solution (Molecular Probes, Eugene, OR). For membrane staining, the HEK-293A cells were washed twice with 1X PBS, fixed in 4% PFA, permeabilized in 0.1% Triton X-100 for 3 min, and washed again in 1X PBS. Cells were incubated in blocking buffer (1% bovine serum albumin [BSA] in PBS) for 10 min followed by Alexa Fluor™ 633 Phalloidin (1:50 dilution in PBS; Molecular Probes) for 20 min and washed twice with 1X PBS before being imaged using a 63X oil objective on a Leica SP8 confocal microscope (Buffalo Grove, IL).