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. 2018 May 24;8(13):3437–3460. doi: 10.7150/thno.23853

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In vitro co-localization with lysosomes and enhanced retention of PC (left) and P-GFLG-Cy5 (right) Turn-ON probes. (A) Representative fluorescence imaging of living 131/4-5B1 melanoma and 4T1 cells treated by LysoTracker® Green (green), a lysosome marker, and Turn-ON probes (red), PC (left) and P-GFLG-Cy5 (right), 2 h post incubation. (B) Analysis of imaged living cells showed 82.1% and 65.7% co-localization (dot plot - orange) of the lysosome marker, LysoTracker® Green, with PC (left) and P-GFLG-Cy5 (right), respectively. Yellow indicates cells with green (Lysotracker) and red (suitable Turn-ON probe) staining (without co-localization), orange indicates co-localization and green indicates non-gated cells. Similarity signal intensity that represents the co-localization of cells with co-localization is presented in the table. (C-D) The cellular uptake of Turn-On probes is higher than that of free Cy5: Internalization of (C) PCQ and (D) P-GFLG-Cy5 probes into live 131/4-5B1 melanoma and 4T1 murine mammary adenocarcinoma cells. Live cells were monitored 1 h following treatment with free Cy5 and the probes. Untreated cells served as control. Brightfield and fluorescence images of (C) PCQ uptake by 131/4-5B1 cells and (D) P-GFLG-Cy5 uptake by 4T1 cells. (E-F) Mean cell fluorescence intensity (blue columns) and percentage of positive cells with Cy5 signal (gray columns) upon (E) PCQ uptake and upon (F) P-GFLG-Cy5 uptake. Free Cy5 uptake and untreated cells served as control in both cell lines. All fluorescence images were obtained using an ImageStream®^X Mark II Imaging Flow Cytometer and analyzed by the supplier software. The data are presented as mean ± SD (n = 3).