Cytosolic mCherry protein expression and transfection
efficiency
4-4-6 and PEI nanoparticles encapsulating the CAG-mCherry plasmid
(Addgene #108685, (Mishra et al., 2020)) were formed as described above
and added to HEK or CHO cells diluted for transfection (2 mL culture in
6-well plate). Optimized DNA concentrations were determined based on
mean fluorescence over 5 days (580± Ex/630Em) using a BioTek SynergyMX
plate reader (BioTek Instruments, Inc.) by reading a 3 × 3 grid
distributed across the well surface area. For transfection efficacy
studies, HEK or CHO cells were dosed with PBAE or PEI nanoparticles
formed as described above, delivering 2 µg/mL (HEK) or 4 µg/mL (CHO)
mCherry DNA (n =5). Cells were collected on day 5 and CHO cells
were treated with Accutase (Sigma Aldrich), according to manufacturer’s
instructions. Cells were then resuspended in phosphate-buffered saline
(PBS, pH 7.4) containing 2% fetal bovine serum (FBS) and stained with
LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen), according to
manufacturer’s instructions. mCherry expression in live cells was
assessed using an Attune NxT Flow Cytometer (ThermoFisher Scientific).
Experiments were performed a minimum of twice with consistent results.