Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

Fuller, Kathryn M.; Arriaga, Edgar A.

doi:10.1016/j.jchromb.2004.03.050

Cited by 27 publications

(37 citation statements)

References 45 publications

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“…Although two different mitochondrial types is not enough to make a general conclusion regarding the N UT for all mitochondria, or any other particles analyzed via CE, previous reports on different kinds of mitochondria show relatively small standard deviations in the quartiles when the samples contain at least 150 events [8,20,30]. Therefore, as practical advice, if a sample of mitochondria is to be analyzed by CE without first identifying the N UT , the number of detected events should exceed 150.…”

Section: Effects Of Undersampling On the Electrophoretic Mobility Quamentioning

confidence: 98%

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Whiting

Arriaga

2007

Journal of Chromatography A

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The number of particles in a sample heavily influences the shape of the distribution describing the corresponding individual particle measurements. Selecting an adequate number of particles that prevents biases due to sample size is particularly difficult for complex biological systems in which statistical distributions are not normal. Quantile analysis is a powerful statistical technique that can rapidly compare differences between multiple distributions of individual particles. This report utilizes quantile analysis to show that the number of events detected affects the mobility distributions for rat liver and mouse liver mitochondria, sample individual particles, when analyzed via capillary electrophoresis with laser-induced fluorescence. When the mitochondrial sample is small (e.g. <78), there are not enough events to obtain statistically relevant mobility data. Adsorption to the capillary surface also significantly affects the mobility distribution at a small number of events in uncoated and dynamically coated capillaries. These adsorption effects can be overcome when the mitochondrial load on the capillary is sufficiently large (i.e. >609 and >1426 events for mouse liver on uncoated capillaries and rat liver on dynamically coated capillaries, respectively). It is anticipated that quantile analysis can be used to study other distributions of individual particles, such as nanoparticles, organelles, and biomolecules, and that distributions of these particles will also be dependant on sample size.

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Section: Effects Of Undersampling On the Electrophoretic Mobility Quamentioning

confidence: 98%

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Whiting

Arriaga

2007

Journal of Chromatography A

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“…This limits hydrophobic interactions between the mitochondria and the fused-silica surface and significantly reduces the EOF. Because AAP-coated capillaries have seen considerable use in the past for the separation of mitochondria on CE, and are one of the gold standards for limitation of mitochondrial, protein, and other organelle adsorption to the fused-silica surface [14,15,23,24,26,28,29], AAP-coated capillaries are used in this report as a reference.…”

Section: Carryover On Aap-coated Capillariesmentioning

confidence: 99%

“…Both flow cytometry and CE show the extraordinary heterogeneous nature of mitochondrial populations [17,18]. Compared to flow cytometry, however, CE-LIF has the analytical advantage of gathering electrophoretic mobility information on particles [19][20][21][22], and consequently, has the potential use in the evaluation of the surface charge and size characteristics of individual mitochondria [14,18,23,24].…”

Section: Introductionmentioning

confidence: 99%

“…In all reported cases of individual mitochondrial analysis on CE [14,15,18,23,24] the fused silica capillary is coated with a layer of the hydrophilic polymer poly(acryloylaminopropanol) (AAP), while the one reported case of mitochondrial analysis on glass microfluidic devices [25] passivated the surface with BSA. Coated capillaries have been used to minimize adsorption of proteins [26], biological particles [14,20,[27][28][29], and liposomal preparations [19][20][21][22] to the fused-silica surface.…”

Section: Introductionmentioning

confidence: 99%

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CE-LIF analysis of mitochondria using uncoated and dynamically coated capillaries

Whiting

Arriaga

2006

Electrophoresis

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This report is the first demonstration of the use of uncoated and dynamically coated capillaries for the separation of individual mitochondria via CE. Currently, the analysis of individual mitochondria relies upon fused-silica capillaries coated with a hydrophilic polymer (e.g. poly(acryloylaminopropanol)), which is used to minimize adsorption to the capillary surface. Both uncoated fused-silica capillaries and 0.2% w/w poly(vinyl alcohol) dynamic coating solutions are used to successfully analyze isolated individual mitochondrial particles using CE-LIF. While it was possible to separate mouse liver mitochondria on an uncoated capillary, rat liver mitochondria proved to have strong adsorption characteristics that only allowed them to be adequately separated with a PVA dynamic coating or a poly(acryloylaminopropanol) (AAP) capillary. The possible causes for this adsorption are analyzed and discussed. This study shows that uncoated and dynamically coated capillaries can be used in place of AAP-coated capillaries to analyze mitochondria and suggests the use of these capillaries for the analysis of other organelles, offering a greatly simplified method for the analysis of individual organelles.

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“…Currently, it is quite common to find microfluidic epifluorescence with LODs of zmol or better [21][22][23][24]. However, these detectors may not be fully adequate in mitochondrial analyses in which there are a significant number of events occurring very close to the LOD [25,26]. Improved LIF detectors, with LODs similar to CE with post-column sheath flow LIF, should be used when analyzing isolated individual mitochondrial events via LIF.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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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 6 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.

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Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

Cited by 27 publications

References 45 publications

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

CE-LIF analysis of mitochondria using uncoated and dynamically coated capillaries

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

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