Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells.

Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.

Continuous organelle separation in an insulator-based dielectrophoretic device.

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 µ$\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size.

Targeting senescent cells alleviates obesity-induced metabolic dysfunction.

Adipose tissue inflammation and dysfunction are associated with obesity-related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug-inducible "suicide" genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra-abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity-related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity-related metabolic dysfunction and its complications.

Fisetin is a senotherapeutic that extends health and lifespan.

BACKGROUND: Senescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity. METHODS: A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated. FINDINGS: Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan. INTERPRETATION: The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies. FUND: NIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/American Federation for Aging Research (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401-1 (JLK, EAA).

Metabolic Labeling with an Alkyne-modified Isoprenoid Analog Facilitates Imaging and Quantification of the Prenylome in Cells.

Protein prenylation is a post-translational modification that is responsible for membrane association and protein-protein interactions. The oncogenic protein Ras, which is prenylated, has been the subject of intense study in the past 20 years as a therapeutic target. Several studies have shown a correlation between neurodegenerative diseases including Alzheimer's disease and Parkinson's disease and protein prenylation. Here, a method for imaging and quantification of the prenylome using microscopy and flow cytometry is described. We show that metabolically incorporating an alkyne isoprenoid into mammalian cells, followed by a Cu(I)-catalyzed alkyne azide cycloaddition reaction to a fluorophore, allows for detection of prenylated proteins in several cell lines and that different cell types vary significantly in their levels of prenylated proteins. The addition of a prenyltransferase inhibitor or the precursors to the native isoprenoid substrates lowers the levels of labeled prenylated proteins. Finally, we demonstrate that there is a significantly higher (22%) level of prenylated proteins in a cellular model of compromised autophagy as compared to normal cells, supporting the hypothesis of a potential involvement of protein prenylation in abrogated autophagy. These results highlight the utility of total prenylome labeling for studies on the role of protein prenylation in various diseases including aging-related disorders.

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics.

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics. Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study, we evaluated the chronic effects of TNFα, IL-6, and IL-1ß on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics, including increased proton leak, decreased ΔΨm, increased basal respiration, and decreased ATP turnover. In contrast, although IL-6 and IL-1ß decreased maximal respiratory capacity, they had no effect on ΔΨm and varied effects on ATP turnover, proton leak, or basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis, including PGC-1α and eNOS as well as deceased expression of COX IV and Cyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation and decreased the total level of OPA1 while increasing OPA1 cleavage, without expression of levels of mitofusin 2, DRP-1, or mitofilin being affected. In summary, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism, but each leads to decreased mitochondrial function and a reprogramming of fat cell biology.

Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies.

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94% of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ∼2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.

Monitoring subcellular biotransformation of N-L-leucyldoxorubicin by micellar electrokinetic capillary chromatography coupled to laser-induced fluorescence detection.

Development of prodrugs is a promising alternative to address cytotoxicity and nonspecificity of common anticancer agents. N-L-leucyldoxorubicin (LeuDox) is a prodrug that is biotransformed to the anticancer drug doxorubicin (Dox) in the extracellular space; however, its biotransformation may also occur intracellularly in endocytic organelles. Such organelle-specific biotransformation is yet to be determined. In this study, magnetically enriched endocytic organelle fractions from human uterine sarcoma cells were treated with LeuDox. Micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF) was used to determine that 10% of LeuDox was biotransformed to Dox, accounting for ~43% of the biotransformation occurring in the post-nuclear fraction. This finding suggests that endocytic organelles also participate in the intracellular biotransformation of LeuDox to Dox.

Evaluation of prenylated peptides for use in cellular imaging and biochemical analysis.

Protein prenylation involves the addition of a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of approximately 2 % of all mammalian proteins. This hydrophobic modification serves to direct membrane association of the protein. Due to the finding that the oncogenic protein Ras is naturally prenylated, several researchers have developed inhibitors of the prenyltransferase enzymes as cancer therapeutics. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation in living cells. Using a combination of flow cytometry and confocal microscopy, we have shown that synthetic fluorescently labeled prenylated peptides enter a variety of different cell types. Additionally, using capillary electrophoresis we have shown that these peptides can be detected in minute quantities from lysates of cells treated with these peptides. This method will allow for further study of the enzymology of protein prenylation in living cells.

Protein carbonylation and metabolic control systems.

Oxidative stress is linked to the production of reactive lipid aldehydes that non-enzymatically alkylate cysteine, histidine, or lysine residues in a reaction termed protein carbonylation. Reactive lipid aldehydes and their derivatives are detoxified via a variety of phase I and phase II systems, and when antioxidant defenses are compromised or oxidative conditions are increased, protein carbonylation is increased. The resulting modification has been implicated as causative in a variety of metabolic states including neurodegeneration, muscle wasting, insulin resistance, and aging. Although such modifications usually result in loss of protein function, protein carbonylation may be regulatory and activate signaling pathways involved in antioxidant biology and cellular homeostasis.

Analysis of the bioactivity of magnetically immunoisolated peroxisomes.

Peroxisomes produce reactive oxygen species which may participate in biotransformations of innate biomolecules and xenobiotics. Isolating functional peroxisomes with low levels of contaminants would be a useful tool to investigate biotransformations occurring in these organelles that are usually confounded with biotransformations occurring in other co-isolated organelles. Here, we immunoisolate peroxisomes and demonstrate that the impurity level after isolation is low and that peroxisomes retain their biological activity. In this method, an antibody targeting a 70-kDa peroxisomal membrane protein was immobilized to silanized magnetic iron oxide beads (1-4 µm in diameter) coated with Protein A. Peroxisomes from L6 rat myoblast homogenates were magnetically captured, washed, and then analyzed for subcellular composition using enzymatic assays. Based on the ratio of peroxisomal to lysosomal activity, the retained fraction is 70-fold enriched relative to the unretained fraction. Similarly, the ratio of peroxisomal activity to mitochondrial content suggests that the retained fraction is >30-fold enriched relative to the unretained fraction. H(2)O(2) production from the ß-oxidation of palmitoyl-CoA demonstrated that the isolated peroxisomal fraction was biologically active. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) analysis confirmed that the immunopurified fractions were capable of transforming the anticancer drug doxorubicin and the fatty acid analog, BODIPY 500/510 C1C12. Besides its use to investigate peroxisome biotransformations in health and disease, the combination of magnetic immunoisolation with CE-LIF could be widely applicable to investigate subcellular-specific biotransformations of xenobiotics occurring at immunoisolated subcellular compartments.

Combination of micellar electrokinetic and high-performance liquid chromatographies to assess age-related changes in the in vitro metabolism of Fischer 344 rat liver.

The metabolism of doxorubicin, a widely used anticancer drug, is different in young adult and old cancer patients. In this study, we demonstrate that micellar electrokinetic chromatography with laser-induced fluorescence detection is highly suited to monitor the metabolism of doxorubicin in subcellular fractions isolated from young adult (11 months, 100% survival rate) and old (26 months, ~25% survival rate) Fischer 344 rat livers. The relative amounts of doxorubicin metabolized in both mitochondria-enriched and postmitochondria fractions of young adult were larger than the respective fractions of old rat liver. 7-Deoxydoxorubicinolone and 7-deoxydoxorubicinone were identified using internal standard addition and structural elucidation by high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection. Although high-performance liquid chromatography with combined laser-induced fluorescence and mass spectrometry detection is more useful in the identification of compounds, micellar electrokinetic chromatography with laser-induced fluorescence detection has low-sample requirements, simplified sample processing procedures, short analysis times and low limit of detection. Therefore, the combination of these two techniques provides a powerful approach to investigate metabolism of fluorescent drugs in aging studies.

Tandem laser-induced fluorescence and mass spectrometry detection for high-performance liquid chromatography analysis of the in vitro metabolism of doxorubicin.

Structural characterization, identification, and quantification of xenobiotics and their metabolic products commonly require the use of at least two different techniques. This has been the case in the analysis of metabolic products of doxorubicin, a widely used fluorescent anthracycline for the treatment of tumors and leukemia. In this work, we combine high-performance liquid chromatography (HPLC) with a tandem laser-induced fluorescence (LIF) and mass spectrometry (MS) detection scheme for the characterization of doxorubicin and its metabolites produced in the postmitochondrial fraction prepared from Fischer 344 rat liver. LIF detection allowed quantification of the metabolic compounds while MS detection aided in the identification of the metabolites. Using this HPLC-LIF-MS methodology, the disappearance of doxorubicin and the appearance of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone were monitored over the course of 40 min. This application demonstrates the potential of the tandem LIF-MS detection scheme in quantification and characterization of biotransformations of fluorescent xenobiotics of biomedical and environmental relevance. Furthermore, this detection scheme would be particularly relevant in the analysis of fluorescent analytes in complex samples and in validation of methods for the analysis of such samples that typically rely only on LIF detectors.

Downregulation of adipose glutathione S-transferase A4 leads to increased protein carbonylation, oxidative stress, and mitochondrial dysfunction.

OBJECTIVE: Peripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function. RESEARCH DESIGN AND METHODS: GSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis. RESULTS: GSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-alpha treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion. CONCLUSIONS: These results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.

Direct sampling from human liver tissue cross sections for electrophoretic analysis of doxorubicin.

After chemoembolization of the liver with doxorubicin (Dox), this drug and its metabolites are not homogeneously distributed in this organ. The distribution cannot be easily measured making it difficult to assess how the drug performs in different tissue regions. Here we report a technique for sampling tissue cross sections that can analyze the contents of micrometer size regions. The tissue cross sections were from the explanted liver of a hepatocellular carcinoma patient. Samples were directly aspirated from a 5 microm thick tissue cross section into a 50 microm i.d. capillary where the tissue was solubilized with a separation buffer containing sodium dodecyl sulfate. Upon sample dissolution, Dox and natively fluorescent compounds were separated and detected by micellar electrokinetic chromatography with laser-induced fluorescence detection. Sampling reproducibility and recovery were assessed using 10% (w/v) gelatin as tissue mimic. Sampling from gelatin slices containing Dox revealed a relative standard deviation of 13%, which was comparable to that of sampling from solution. Dox recovery was 82% +/- 16% (n = 5). When sampling tumor and nontumor tissue regions, samples could be taken from the same region 100 microm apart. Atomic force microscopy was used to determine that each sample was 8.4 +/- 1.0 pL in volume which made it possible to determine Dox concentrations in the ranges of 0.4-1.3 and 0.3-0.5 microM for the samples taken from tumor and nontumor regions, respectively. The results demonstrated the feasibility of sampling, detection, and quantification of Dox in micrometer size regions, which could be a useful resource for analyzing the Dox concentration and distribution in highly heterogeneous tissues.

Qualitative determination of superoxide release at both sides of the mitochondrial inner membrane by capillary electrophoretic analysis of the oxidation products of triphenylphosphonium hydroethidine.

Superoxide is released asymmetrically to both sides of the mitochondrial inner membrane. Because this membrane is impermeable to superoxide, two separate pools are formed at either side of the membrane, each with its own characteristics and potential biological effects. Here, we report an attomole-sensitive fast capillary electrophoretic method that can analyze superoxide in a single pool, either the matrix pool or that outside the mitochondria. The method uses triphenylphosphonium hydroethidine, which reacts with the superoxide in both pools. Centrifugation is used to separate the mitochondria (i.e., matrix contents) from the supernatant (i.e., products released outside the mitochondria). Each fraction is then analyzed by capillary electrophoresis with laser-induced fluorescence detection that separates and detects hydroxytriphenylphosphonium ethidium (OH-TPP-E+), the fluorescent superoxide-specific product. The separation takes <3 min and the detection level is down to 3 amol OH-TPP-E+. The method has proved to be effective at detecting superoxide release qualitatively in the mitochondria of 143B cells, mouse liver, and rat skeletal muscle, in both the presence and the absence of inhibitors. In addition, this study confirmed that complex I releases superoxide only toward the matrix, whereas complex III releases superoxide toward both sides of the mitochondrial inner membrane. Furthermore, treatment with menadione induces superoxide release toward both sides of the mitochondrial inner membrane.

Determining under- and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser-induced fluorescence detection.

This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 +/- 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile-quantile (q-q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (>72 for rat liver and >98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q-q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (<55 for rat liver and <81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.

Monitoring incorporation, transformation and subcellular distribution of N-l-leucyl-doxorubicin in uterine sarcoma cells using capillary electrophoretic techniques.

Previous reports have demonstrated that N-l-leucyl-doxorubicin (LeuDox) is less toxic than its parent drug, Dox, but the underlying causes of this reduced toxicity have yet to be fully elucidated. In this study, the incorporation of LeuDox into (i) the MES-SA human uterine sarcoma cell line and (ii) its Dox resistant counterpart, MES-SA/Dx5 cell line and the subsequent transformation of LeuDox into Dox and its subcellular distribution, were investigated by micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF). In both cell lines the cellular uptakes of Dox and LeuDox were similar at equimolar doses, while the percent transformation of LeuDox into Dox in MES-SA/Dx5 cells was about twice as great as its transformation in MES-SA cells, which is beneficial for reaching Dox cytotoxic levels in this resistant cell line. When both cells lines were treated with IC(35) concentrations of either Dox and LeuDox, the intracellular Dox amounts were 6-fold higher in the resistant cell line than in the sensitive cell line, suggesting that other cellular processes play a role in the cytotoxicity of Dox in the resistant cell line. The amounts and ratios of Dox and LeuDox in four subcellular fractions of LeuDox-treated MES-SA/Dx5 cells were also investigated. The highest Dox/LeuDox ratio (i.e. 2.92) was found in the nuclear fraction, followed by the ratio in the low density organelle fraction (i.e. 1.92) that contains lysosomes, organelles in which lysosomal hydrolytic enzymes, capthesins, transform LeuDox into Dox.

Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis.

Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.

CE analysis of the acidic organelles of a single cell.

The properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles.