Cellular colocalization of TM601-488 and Lyso-Tracker-Red DND 99 by direct fluorescence. Images of live cells and cellular colocalization of TM601-488 (green) and Lyso-Tracker-Red DND 99 (red) detected by direct fluorescence in NHDF normal human fibroblasts (A), NHA normal human astrocytes (B), and U373 human glioma (C) using the Colocalization Tool (as described in the Methods). An overlay mask (white pixels) placed on each image visualizes the colocalization of pixels above the background threshold showing the highest level of white pixels in the U373 glioma (C). The following three fluorescent color channels were used: green (TM601-488), red (Lyso-Tracker- Red), and yellow (merge of red and green fluorescence) to determine the extent of colocalization of TM601 and LysoTracker by confocal microscopy. The merge red and green fluorescence in glioma cells (F) was compared to the controls of TM601-488 (D) alone or LysoTracker Red (E) alone. The images in D, E, and F are representative of n = 7 experiments. The Colocalization Tool in A, B, C was used in one out of 7 experiments evaluating total of 14-29 cells in 3 fields. Scale bars = 10 μm, magnification 630 ×.