Astrios eq sorter

NIH3T3
cells were exposed to one of the following conditions for 30 min prior
to transfection: (1) incubated at 4 °C (as opposed to 37 °C)
or (2) addition of 0.45 M sucrose (Sigma, S9378),^{21 (link),32 (link)} (3) 100 μM 5(N-ethyl-N-isopropyl)
amiloride (EIPA) (Sigma, A3085),^{33 (link)} and
(4) 5 mM methyl-B-cyclodextrin (MBCD) (Sigma, C4555)^{34 (link),35 (link)} in growth medium. Cells were transfected with FLR-DNA or FLR-DNA-MNPs
(formulated with Rh-pDNA) and incubated for 1 h with or without exposure
to a magnetic field. Transfection was carried out at 4 °C for
inhibition at low temperature. All other transfections were carried
out at 37 °C. The control group was transfected at 37 °C
in growth medium without inhibitors. After one hour, cells were washed
with PBS or heparin (100 μg/mL). Red fluorescence in the cells
was quantified by flow cytometry. Each sample was run individually
through a flow cytometer; 50,000–100,000 total events were
recorded per sample (Astrios EQ sorter, Beckman Coulter, US). Untreated
cells were used as a control.

GFP-expressing
cells were imaged by fluorescence (Leica DM IRB). GFP transfection
efficiency (% positivity) and expression intensity were quantified
by flow cytometry. Total events (50,000–100,000) were recorded
per sample (Astrios EQ sorter, Beckman Coulter, US). Untreated cells
were used as a control. For targeting within a single culture, cells
were plated as a contiguous monolayer within wells of a 6-well plate
(34.8 mm diameter, 9.5 cm² culture area) containing a sterile
coverslip (borosilicate glass, 20 mm diameter, and 3.1 cm² culture area). Coverslips were affixed to the center of the culture
surface with sterile vacuum grease, allowing them to be readily removed
with forceps after seeding, exposure, and washing. On transfection,
targeting to the coverslip was achieved by placing the well on the
array as previously described, a 20 mm diameter magnet aligning with
the coverslip. After the incubation, the array was removed, and cells
were washed as described before with PBS or heparin. The coverslip
was removed to a fresh well with forceps, and targeted (IN region,
3.1 cm²) and untargeted (OUT region, 6.4 cm²) cells were incubated as before analysis.