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. Author manuscript; available in PMC: 2013 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2012 Jul 3;1820(11):1734–1743. doi: 10.1016/j.bbagen.2012.06.020

Figure 5. Co-incubation of TMR-TAT with the cell-impermeable calcium chelator EGTA inhibits cell death induced by TM-PCI.

Figure 5

A) HeLa cells were incubated with TMR-TAT (3 μM) in media containing calcium (1.3 mM) with or without EGTA (2 mM) for 30 min. Cells were then washed and imaged in media containing calcium (1.3 mM) to avoid calcium depletion of intracellular organelles. As EGTA is cell-impermeable, it is expected to accumulate inside endocytic organelles with TMR-TAT. Fluo-4 imaging indicates that TMR-PCI did not cause a significant increase in fluo-4 signal as compared to the control without EGTA present. B) Fluorescence imaging of cells incubated with EGTA and TMR-TAT after exposure to light for 6 sec. The intensity of the fluorescence signals of TMR-TAT inside cells indicate that incubation with EGTA does not cause a significant reduction in the amount of TMR-TAT endocytosed and released into the cytosol after light irradiation. C) Quantitative analysis of cell killing that accompanies TM-PCI in cells incubated without or with EGTA. A SYTOX® Green exclusion assay was used to distinguish live cells (stained by Hoechst, pseudo-colored purple) from dead cells (stained by SYTOX® Green, pseudo-colored green). The histogram represents the fraction of cells stained by SYTOX® Green before and after light irradiation. The average values of three experiments and the corresponding standard deviations are presented along with two representative SYTOX® Green and Hoechst overlay microscopy images.