Cells were seeded
in a 24-well plate (100,000 cells per well) with 0.5 mL culture media
and incubated for 24 h. A solution of Alexa-488 labeled compounds
(75 μL, 1.5 mg mL
–1) in culture media was
dosed to the cells in triplicate under three experimental conditions:
incubation at 37 °C for 3 or 24 h and incubation at 4 °C
for 3 h. For the 4 °C experiment, the 24-well plate and sample
solution were cooled on ice for 10 min prior to the dosage of the
compound and kept on ice for the duration of incubation. After incubation,
the culture media was then removed, and the cells were washed twice
with cold PBS, treated with Trypsin, and incubated for 10 min to harvest
them. A solution of bovine serum albumin (BSA, 10%, 0.3 mL) was added
to each well, transferred to a 96-well plate, and centrifuged at 350
G for 5 min, after which the supernatant was discarded, and the cells
were resuspended in 10% BSA solution. Samples were analyzed using
a
S100EXi flow cytometer (Stratedigm), equipped with 405, 488, 552,
and 640 nm solid-state lasers. Forward and side scatter gates were
used to exclude debris and dead cells using a viability dye (propidium
iodide). The mean fluorescence intensity for a population of 10,000
cells was determined with Flowjo v8 for each experimental condition
in triplicate.
Kerr A., Sagita E., Mansfield E.D., Nguyen T.H., Feeney O.M., Pouton C.W., Porter C.J., Sanchis J, & Perrier S. (2022). Polymeric Nanotubes as Drug Delivery Vectors—Comparison of Covalently and Supramolecularly Assembled Constructs. Biomacromolecules, 23(6), 2315-2328.