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.

Inhibition of Macroautophagy Triggers Apoptosis

“…HeLa cells exposed to the lysosomotropic agent HCQ manifested a transiently increased level of staining with LTR (LTR high cells) (Fig. 1A); LTR is a fluorochrome which measures the volume of the acidic vacuolar compartment (14,18). This increased LTR labeling was observed well before the ⌬⌿ m [as measured with DiOC 6 (3), a ⌬⌿ m -sensitive dye] dropped, before PS residues were exposed on the plasma membrane surface (quantified with an annexin V-fluorescein isothiocyanate conjugate), and before irreversible plasma membrane permeabilization (determined with PI) occurred (Fig.…”

Capillary electrophoresis of biological particles: Viruses, bacteria, and eukaryotic cells

“…The mobility distribution (ranging between 12 and 43610 29 m 2 V 21 s 21 ) was dependent on the kind of cells from which the mitochondria were isolated. The same group determined also the mobility distribution of individual acidic organelles of NS-1 mouse myeloma cells [45]. The organelles were labeled with fluorescent microspheres (with 0.1 mm diameter), which were internalized into the cells via endocytosis, allowing for detection of the organelles with LIF.…”

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

“…Although flow cytometry was historically used first to detect individual organelles [9,10], CE has the additional analytical advantage of being able to separate organelles based on their electrophoretic mobilities [11][12][13][14]. In general, the electrophoretic mobility of a particle stems from its size [12,14,19] and surface characteristics [13,19], which makes CE useful for comparing the size or surface characteristics of individually detected biological species, such as proteins [15], mitochondria [8,16], nuclei [17], and acidic organelles [18]. The size and surface characteristics of these particles may be highly heterogeneous, making it extremely important to analyze samples with a sufficient number of individual species so that an accurate statistical description is observed.…”