Luciferase and BCA Assay. Prior to transfection, HDFn cells (Invitrogen) were plated on 24-well plates at a density of 100,000 cells per well, approximately 70% confluency. Cells were incubated in Medium 106, supplemented with 2% FBS, hydrocortisone (1 µg/mL), human epidermal growth factor (1 ng/mL), basic fibroblast growth factor (3 ng/mL), and heparin (10 µg/mL), for 24 hours at 37ºC in a 5% CO2 environment.
Cells were starved of sera 30 minutes prior to transfection. Transfection reagents (two leading lipid reagents and Glycofect) were formulated with pDNA based upon their recommended protocols. Solutions of transfection reagent-pDNA (gWiz-Luciferase, Aldevron) complexes for each transfection reagent were added in triplicate to corresponding wells (1 µg pDNA per well). Plates were briefly swirled and incubated for four hours, after which more media (500 µL) was added to each well. Cells were incubated for an additional 20 hours, followed by a media change and 24 hours of additional incubation time. Media was evacuated from wells and cells were lysed in 100 µL Cell Lysis Buffer (Promega).
Cell lysates were deposited on 96-well plates and analyzed for luciferase activity on a luminometer plate reader (Tecan GENios Pro). Protein lysates were stained using a BCA staining procedure, and their concentration was determined with absorbance measurements at 750 nm. Cell viability was determined by sample absorbance relative to the “cells only” control.
MTT Assay. Cells were prepared and transfected using the same methodology as for the luciferase and BCA assays. However, at the 47 hour time point media was evacuated from each well and replaced with media containing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, [MTT] = 0.5 mg/mL). Cells were incubated for an additional 1 hour then washed with PBS and lysed in 250 µL DMSO. Sample cell lysates were analyzed via absorbance vs. cells only lysate’s absorbance to determine cell viability.
GFP Analysis via Flow Cytometry. Cells were plated on 6-wells and polyplexes were formed using the same methods reported using a plasmid-encoding enhanced green fluorescent protein (EGFP-C1, 5µg total per well). Transfection and media change conditions are consistent with those reported above. After 48 hours cells were trypsinized, washed with PBS twice, and suspended in 500 µL PBS. Flow cytometry analysis of each sample provided mean fluorescence intensity as well as the percentage of cells positive for EGFP.
GFP Analysis via Fluorescent Microscopy. Cells were plated using the same methods performed for the flow cytometry experiment. Transfection and media change conditions are also consistent with those reported above. After 48 hours, cells were washed with PBS twice and fixed in a 4% para-formaldehyde PBS buffer solution. Cells were imaged on a Nikon TE2000U fluorescent microscope for both differential interference contrast images and for GFP florescence (ex. 488 nm, em. 509 nm).