Table 1.

Summary of probes commonly utilized to assess lysosomal pH

Probe	Usage	Excitation/Emission	pKa	pH optima	Incubation time*	Limitations
Lysosensor Yellow/Blue- Dextran	in vitro	384nm/440nm 540nm	~4.2	3–9	12–24 hours	Weak fluorescence if dye concentrations are not titrated correctly
Fluorescein- Dextran	in vitro	494nm/521nm	~6.2	5–8	12–24 hours	Fluorescent intensity minimal in lysosomal pH range, Optimal for early and late endosomal quantification
Lysosensor Yellow/Blue DND-160	in vitro	329nm/440nm 540nm	~4.2	3–9	5 min maximum	Accumulates in all acidic compartments no lysosomal selectivity Incubation longer than 5 minutes results in compartment alkalinization
pHrodo- Dextran	in vitro	560nm/585nm	N/A	5–8	12–24 hours	Fluorescent intensity increases with acidity Lack of utilization in literature Lack of dual excitation/emission means it must be utilized in conjunction with a pH insensitive dye of different wavelength.
Lysotracker Red-99	in vitro	577nm/590nm	N/A	< 5.2	30 min	Qualitative assessment only Accumulates in all acidic compartments Should not be sole method for assaying lysosomal pH
DAMP	in vivo	N/A	N/A	N/A	12 hours in anti-DNP primary antibody	Qualitative assessment only Requires difficult immunogold EM protocols Must be injected directly into tissue to be studied Accumulates in all acidic compartments although specific compartments can me assessed at EM level

^*Incubation times should be empirically determined for individual cell lines