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.