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

Whiting, Christofer E.; Arriaga, Edgar A.

doi:10.1002/elps.200600249

Cited by 18 publications

(36 citation statements)

References 37 publications

(59 reference statements)

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“…While the electrophoretic mobility histograms also appear similar to the previous CE reports [17,28], it can be difficult to make a detailed comparison of a large number of distributions using histograms, particularly when these distributions cannot be easily mathematically described. Thus, a better method for comparing mobility distributions was developed for this purpose.…”

Section: Identifying Sampling Effects On Mitochondrial Distributionsmentioning

confidence: 60%

“…2, which resembles the electropherograms of individual mitochondrial previously obtained on CE [17,28] and microfluidic devices [15]. In order to prevent unwanted adsorption to the glass microfluidic device, a buffer containing 0.2% w/w PVA was used.…”

Section: Identifying Sampling Effects On Mitochondrial Distributionsmentioning

confidence: 93%

“…This centrifugation step was repeated three times. The remaining mitochondrial fraction was then frozen and stored in liquid nitrogen as described previously [28].…”

Section: Mitochondrial Preparationmentioning

confidence: 99%

“…Table 2 compares the average 0.25, 0.50, and 0.75 quantiles for the unbiased microfluidic distributions and CE distributions of rat liver mitochondria separated under identical conditions [28]. A t-test shows that these quantiles are not statistically different (p = 0.05), suggesting that microfluidic devices are comparable to CE for the determination of mitochondrial electrophoretic mobility distributions, if oversampling and undersampling can be avoided.…”

Section: Unbiased Distributionsmentioning

confidence: 99%

“…The two types of mitochondria chosen for analysis in this report were isolated from rat liver tissue and cultured 143B cells, an osteosarcoma cell line. Rat liver mitochondria were chosen because they represent a difficult separation in CE due to their high degree of adsorption to the fused-silica surface [28], while mitochondria from 143B cells were chosen because the dependence of their electrophoretic mobility distribution on sample size has not yet been studied.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Identifying Sampling Effects On Mitochondrial Distributionsmentioning

confidence: 60%

Section: Identifying Sampling Effects On Mitochondrial Distributionsmentioning

confidence: 93%

“…This centrifugation step was repeated three times. The remaining mitochondrial fraction was then frozen and stored in liquid nitrogen as described previously [28].…”

Section: Mitochondrial Preparationmentioning

confidence: 99%

Section: Unbiased Distributionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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Recent advances in the analysis of biological particles by capillary electrophoresis

Košťál

Arriaga

2008

Electrophoresis

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Continuous intact cell detection and viability determination by CE with dual‐wavelength detection

Ren

Zhang

et al. 2010

Electrophoresis

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We introduce here a method for continuous intact cell detection and viability determination of individual trypan blue stained cells by CE with ultraviolet-visible dual-wavelength detection. To avoid cell aggregation or damage during electrophoresis, cells after staining were fixed with 4% formaldehyde and were continuously introduced into the capillary by EOF. The absorbance of a cell at 590 nm was used to determine its viability. An absorbance of two milli-absorbance unit at 590 nm was the clear cut-off point for living and dead Hela cells in our experiments. Good viability correlation between the conventional trypan blue staining assay and our established CE method (correlation coefficient, R(2)=0.9623) was demonstrated by analysis of cell mixtures with varying proportions of living and dead cells. The CE method was also used to analyze the cytotoxicity of methylmercury, and the results were in good agreement with the trypan blue staining assay and 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide methods. Compared with the 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide method, our established CE method can be easily automated to report cell viability based on the state of individual cells. Tedious manual cell counting and human error due to investigator bias can be avoided by using this method.

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

Cited by 18 publications

References 37 publications

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

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

Recent advances in the analysis of biological particles by capillary electrophoresis

Continuous intact cell detection and viability determination by CE with dual‐wavelength detection

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