Figure 3

In vivo pH calibration curve of fluorescein-dextran (F ₅₃₅ (λ ₄₈₅ )/F ₅₃₅ (λ ₄₄₀ ) ratio against pH). Lysosomes of live J774A.1 cells were labeled with fluorescein-dextran and then incubated with MES calibration buffer solutions (pH from 4.0 to 7.0) containing nigericin and monensin. Fluorescence images of emission wavelength at 515–565 nm were acquired under the excitation at 485 and 440 nm (i.e. F₅₃₅(λ₄₈₅) and F₅₃₅(λ₄₄₀), respectively). The ratio of F₅₃₅(λ₄₈₅) over F₅₃₅(λ₄₄₀) in the lysosomes was then calculated as described in the Methods section.