The LysoTracker probes consist of a hydrophobic fluorophore linked to a weak base that is only partially protonated at neutral pH. In a neutrally charge state, the LysoTracker probes can freely diffuse across intact plasma membranes of live cells. Due to the inherently acidic properties within the lysosome, upon diffusion into the lysosome the weakly basic moiety is protonated. In this charged state, the probe does not readily diffuse across the organelle membrane, providing a localized accumulation for distinct staining of acidic organelles such as lysosomes. Effective at nanomolar concentrations, the LysoTracker probes are highly selective for acidic organelles and provide simple one-step staining that does not rely on antibody detection.

The LysoSensor probes are fluorescent pH indicators that partition into acidic organelles. The LysoSensor dyes are acidotropic probes that appear to accumulate in acidic organelles as the result of protonation, which enables researchers to study the dynamic aspects of lysosome biogenesis and function in live cells. This protonation also relieves the fluorescence quenching of the dye by its weak base side chain, resulting in an increase in fluorescence intensity. Thus, the LysoSensor reagents exhibit a pH-dependent increase in fluorescence intensity upon acidification, in contrast to the LysoTracker probes, which exhibit fluorescence that is largely independent of pH.

The lysosomes are acidic in nature due to the production and storage of digestive enzymes (hydrolases). The acidic environment of lysosomes enables degradation of carbohydrates, lipids, nucleic acids, and peptides. Extracellular proteins, virus or bacteria can also be internalized and trafficked to the lysosome for degradation via the lysosomal proteolysis pathway. In addition, misfolded proteins or damaged organelles become targets for lysosomal degradation via the induction of autophagy. Autophagy is important for cellular differentiation, survival during nutrient deprivation, and normal growth control. The inherent acidic nature within lysosomes, and any change of pH during biosynthesis or pathogenesis, can be exploited by various probes that respond to an acidic environment. The LysoTracker and LysoSensor products provide fluorescence detection for live-cell staining of acidic environments and can be used for labeling and tracing acidic organelles such as lysosomes.

Features of the LysoTracker and LysoSensor probes:
LysoTracker Red DND-99 is a cell-permeable red fluorescent dye that stains acidic compartments within a cell, such as lysosomes. LysoTracker Red DND-99 has an excitation and emission maximum of 577/590 nm and can be efficiently excited using a TRITC filter. Although it does not crosslink with cells, the LysoTracker Red DND-99 shows transient retention after methanol-free formaldehyde fixation for a short period of time.

LysoTracker Green DND-26 is a cell-permeable, non-fixable, green fluorescent dye that stains acidic compartments within a cell, such as lysosomes. LysoTracker Green DND-26 has an excitation and emission maximum of 504/511 nm and can be efficiently excited using a FITC filter.

LysoSensor Green DND-189 can be used to measure the pH of acidic organelles, such as lysosomes. This LysoSensor dye becomes more fluorescent in acidic environments. The pKa of the LysoSensor Green DND-189 is ∼5.2.

LysoSensor Yellow/Blue DND-160 is a ratiometric probe that can be used to measure the pH of acidic organelles, such as lysosomes. This LysoSensor dye produces blue fluorescence in neutral environments, but changes to yellow fluorescence in more acidic environments. The pKa of LysoSensor Yellow/Blue DND-160 is ∼4.2.

Spectral characteristics of the LysoTracker and LysoSensor probes:
• LysoTracker Green DND-26—504/511 nm
• LysoTracker Red DND-99—577/590 nm
• LysoSensor Green DND-189—443/505 nm
• LysoSensor Yellow/Blue DND-160—553/570 nm