Individual acidic organelle pH measurements by capillary electrophoresis.
This report describes the pH measurement of individual acidic organelles isolated from the human leukemia CCRF-CEM and CEM/C2 cells. These cells were allowed to endocytose fluorescein tetramethylrhodamine dextran (FRD), a ratiometric probe that has fluorescein as a pH-dependent fluorophore and tetramethylrhodamine as a pH-independent fluorophore. Isolated organelle fractions from these cells were then subjected to capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis. The detection of individual organelle fluorescence at two different wavelengths, selected on the basis of the emission range of the FRD probe, gives a fluorescence intensity ratio used to calculate the pH from a calibration curve. This curve was constructed from CE-LIF measurements of individual liposomes loaded with several pH buffer standards. The respective median pH values are 5.1 +/- 0.2 in CEM/C2 cells and 6.1 +/- 0.4 in CCRF-CEM cells. These measurements compare well with pixel-based epifluorescence microscopy measurements of whole cells where the corresponding average pH values are 5.0 +/- 0.6 (n = 15) and 6.2 +/- 0.7 (n = 15). A pH comparison between the two cell types suggests that the lower pH in the CEM/C2 cells may be relevant to the protonation and sequestration of weak base anticancer drugs such as doxorubicin. The determination of the pH of individual vesicles, liposomes, and acidic organelles is a new resource for measuring and investigating the role of the acid-base properties of subcellular-size compartments.
Chen
Yun
Y
Department of Chemistry, University of Minnesota, Minneapolis, 55455, USA.
Arriaga
Edgar A
EA
D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000379:methods; D015388:Organelles / Q000737:chemistry*; D012680:Sensitivity and Specificity