Analysis of individual acidic organelles by capillary electrophoresis with laser-induced fluorescence detection facilitated by the endocytosis of fluorescently labeled microspheres.

Abstract

Submicrometer-sized fluorescent microspheres were loaded into the acidic organelles of NS-1 mouse myeloma cells via endocytosis. Confocal microscopy imaging showed that microspheres colocalized nearly perfectly with LysoTracker Red, a probe that stains acidic organelles. Unlike LysoTracker dyes that seem to leak from acidic organelles upon cell disruption, microspheres are retained within these organelles, facilitating their analysis following isolation. Using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF), the electrophoretic mobilities of acidic organelles were individually calculated and fluorescence intensities individually measured. When cells were incubated for sufficient time to allow for endocytosis (48 h) with 3.9 x 10(3) microspheres/cell, replicate CE-LIF analyses of the corresponding isolated fraction indicated a dramatic increase in the number of detected events (n = 1990 +/- 234) and in the overall fluorescence intensity of the individual events (0.38 +/- 0.01 RFU; average +/- SD; n = 3) over the corresponding <10-min incubations (n = 60; 0.21 RFU, respectively). In addition, a treatment with 4-fold increase in microsphere density (1.6 x 10(4) microspheres/cell), increased the number of detected individual events (n = 3427 +/- 101) and altered only slightly the fluorescence intensity and electrophoretic mobility distributions. The individual electrophoretic mobility values ranged from -1.45 x 10(-)(4) to -3.0 x 10(-)(4) cm(2) V(-)(1) s(-)(1) while the individual fluorescence values ranged from 0.1 V to over 8 V, demonstrating the benefit of detecting organelles individually rather than averaging their properties over single cells or bulk homogenates.