Flow cytometric measurement of penetratin uptake and
endo-lysosomal release
Cells were incubated in the continuous presence of 5 µM
AFDye532-penetratin, 5 µM NF-penetratin and 0.25 µg/ml DAPI at 37°C in
the thermostated sample holder of a Becton Dickinson FACSAria III flow
cytometer (Becton Dickinson, Mountain View, CA). DAPI was excited at 405
nm, and its emission was measured between 430-470 nm. Both
AFDye532 and NF were excited at
561 nm, and their fluorescence was recorded at 515-545 nm and 663-677
nm, respectively. Apart from background measurements every experiment
was carried out for 20 min and the measurement started right after
adding the DAPI/penetratin mixture, preheated to 37°C, to the cells.
After spectral compensation and gating out dead cells based on DAPI
positivity the time-correlated fluorescence intensities were exported
using FCS Express (De Novo Software, Pasadena, CA). A custom-written
Matlab program was used for calculating a moving average with a window
size of 20 seconds. The pH-insensitive fluorescence of AFDye532 is
proportional to the total cellular uptake of penetratin. Due to
quenching of NF at acidic pH, the ratio of NF-penetratin to AFDye532
fluorescence intensities reports the endo-lysosomal escape of the
cell-penetrating peptide (Qian, Dougherty & Pei, 2015). The
fluorescence intensities of both indicators were normalized to the mean
intensity measured in the first time window. Normalized mean intensities
were divided by each other to obtain the
NF-penetratin/AFDye532-penetratin ratio. The standard error of the mean
(SEM ) of the ratio parameter was calculated using error
propagation analysis assuming independence of the two fluorescence
intensities:
designate the mean intensity of the respective parameter.