In vivo pH calibration curve of fluorescein-dextran (F535(λ485)/F535(λ440) 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.
Chen BMC Cell Biology 2002 3:21 doi:10.1186/1471-2121-3-21