Characterization of a rapid cellular-fractionation technique for hepatocytes. Application in the measurement of mitochondrial membrane potential in situ

A rapid cellular-fractionation technique [Hoek, Nicholls & Williamson (1980) J. Biol. Chem. 255, 1458-1464] was further characterized by using hepatocytes. Of the mitochondrial marker-enzyme activity, 80% was routinely separated from 71-98% of the total cell activities of marker enzymes for plasma membranes, Golgi-membranes, endoplasmic reticulum, lysosomes and cytosol. The mitochondria were contaminated with 53% of cell nuclei. [3H]Triphenylmethylphosphonium ion (TPMP+) was added to hepatocytes in an attempt to measure cellular transmembrane electrical potentials. After rapid cell fractionation the electrical potential between mitochondria in situ and the incubation medium was found to be 202 mV. This value was slightly increased when hepatocytes were treated with oligomycin, but substantially decreased by oligomycin plus an uncoupler of oxidative phosphorylation. Although estimates of TPMP+ binding were obtained, substantial difficulties prevented the accurate measurement of the electrical potential across the plasma membrane. It is concluded that TPMP+ may be employed to demonstrate the integrity of mitochondria during the fractionation procedures. However, the cation is inadequate for the determination of the separate components of the electrical potential between the mitochondrial matrix and the incubation medium.

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Modified ferron assay for speciation characterization of hydrolyzed Al(III): a precise k value based judgment

Water Science & Technology ◽

10.2166/wst.2009.066 ◽

2009 ◽

Vol 59 (4) ◽

pp. 823-832 ◽

Cited By ~ 3

Author(s):

Ye Changqing ◽

Wang Dongsheng ◽

Wu Xiaohong ◽

Qu Jiuhui ◽

John Gregory

Keyword(s):

Kinetic Constant ◽

Nonlinear Least Squares ◽

Precise Determination ◽

K Value ◽

First Order ◽

First Order Kinetics ◽

Parallel Reactions

The speciation of Al-OH complexes in terms of Ala, Alb and Alc could be achieved by traditional ferron assay and Alb is generally considered as Al13, however, the inherent correlation between them remains an enigma. This paper presents a modified ferron assay to get precise determination of Al13 using nonlinear least squares analysis, and to clarify the correlation between Alb and Al13. Two parallel reactions conforming to pseudo-first-order kinetics can simulate the complicate reactions between polynuclear complexes and ferron successfully. Four types of experimental kinetic constant (k value) of Al-OH complexes can be observed by this method when investigating three typical aluminium solutions. Comparing with the results of 27Al NMR, the species with moderate kinetics around 0.001 s−1 can be confirmed to resemble to Al13 polycation. The other types of kinetics are also well-regulated in partially neutralized aluminium solutions with various OH/Al ratios (b values) in the range 0 ∼ 2.5. It would provide potential means to trace the in-situ formation of Al13 in dilute solutions such as coagulation with Al-based coagulants

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In situ structure and force characterization of 2D nano-colloids at the air/water interface

Soft Matter ◽

10.1039/c9sm01476d ◽

2019 ◽

Vol 15 (42) ◽

pp. 8475-8482

Author(s):

Giovanni Li-Destri ◽

Roberta Ruffino ◽

Nunzio Tuccitto ◽

Giovanni Marletta

Keyword(s):

Quantitative Determination ◽

Experimental Method ◽

Water Interface ◽

Liquid Interfaces ◽

Interaction Forces ◽

Air Water Interface

We have developed a novel experimental method, which enables quantitative determination of interaction forces between interfacial nanoparticles as a function of the inter-particle distance at liquid interfaces.

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In situ calibration and [H+] sensitivity of the fluorescent Na+indicator SBFI

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.6.c1623 ◽

2001 ◽

Vol 280 (6) ◽

pp. C1623-C1633 ◽

Cited By ~ 48

Author(s):

Abdoullah Diarra ◽

Claire Sheldon ◽

John Church

Keyword(s):

Ph Dependence ◽

Apparent Dissociation Constant ◽

Simple Method ◽

Standard Equation ◽

In Situ Calibration ◽

Apparent Decrease ◽

Calibration Techniques

Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+]i), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440–3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a “full” in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant ( K d) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a “one-point” technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+]i using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K d of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8–7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+]i values. The magnitudes of the apparent changes in [Na+]ivaried with absolute [Na+]i, and a method was developed for correcting [Na+]i values measured with SBFI for changes in intracellular pH.

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Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.296 ◽

1984 ◽

Vol 4 (2) ◽

pp. 296-301 ◽

Cited By ~ 7

Author(s):

B Storrie ◽

M Sachdeva ◽

V S Viers

Keyword(s):

Horseradish Peroxidase ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Fluid Phase ◽

Ovary Cell ◽

Cell Fractionation ◽

Marker Enzyme ◽

Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

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Advancements in Techniques for Calibration and Characterization of In Situ Optical Particle Measuring Probes, and Applications to the FSSP-100 Probe

Journal of Atmospheric and Oceanic Technology ◽

10.1175/jtech2006.1 ◽

2007 ◽

Vol 24 (5) ◽

pp. 745-760 ◽

Cited By ~ 17

Author(s):

Dagmar Nagel ◽

Uwe Maixner ◽

Walter Strapp ◽

Mohammed Wasey

Keyword(s):

High Speed ◽

Sample Volume ◽

Absolute Calibration ◽

Droplet Generator ◽

Measuring Systems ◽

Distortion Matrix ◽

Measured Particle

Abstract Advancements in techniques for the operational calibration and characterization of instrument performance of the Particle Measuring Systems, Inc. (PMS), forward scattering spectrometer probe (FSSP) and optical array probes (OAPs) are presented, which also can be used for most in situ particle-measuring optical probes on the market. These techniques include the determination of a distortion matrix to correct for instrumental broadening of the measured particle size distribution. A new version of a monodisperse droplet generator is introduced for absolute calibration in the size range between 10 and 100 μm. In addition, a high-speed technique was employed for the determination of airspeed influence on the sample volume and the sizing of particles. The calibration of a PMS FSSP with real water droplets may be significantly different from the usual calibration with glass beads. High-speed measurements simulate particles at speeds of up to about 250 m s−1. Particle undersizing and the decrease of the sample volume with increasing airspeed are described. The use of the modular tools, built for this work, is discussed for probe alignment, functionality checks, and general characterization and diagnostics both in laboratory and field environments.

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Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA.

The Journal of Cell Biology ◽

10.1083/jcb.65.2.309 ◽

1975 ◽

Vol 65 (2) ◽

pp. 309-323 ◽

Cited By ~ 7

Author(s):

R S Tuan ◽

K P Chang

Keyword(s):

Plasma Membranes ◽

Structural Integrity ◽

Light And Electron Microscopy ◽

Heat Denaturation ◽

Marker Enzyme ◽

Genome Complexity ◽

Numerical Abundance ◽

Cellular Components ◽

Isopycnic Centrifugation

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote-containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.

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Resolution Limits of Secondary Electron Dopant Contrast in Helium Ion and Scanning Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927611000365 ◽

2011 ◽

Vol 17 (4) ◽

pp. 637-642 ◽

Cited By ~ 9

Author(s):

Mark Jepson ◽

Xiong Liu ◽

David Bell ◽

David Ferranti ◽

Beverley Inkson ◽

...

Keyword(s):

Secondary Electron ◽

Electrical Potential ◽

Helium Ion ◽

Resolution Limits ◽

Contrast Mechanism ◽

Material Contrast ◽

Dopant Atoms ◽

Scanning Electron

AbstractAs the miniaturization of semiconductor devices continues, characterization of dopant distribution within the structures becomes increasingly challenging. One potential solution is the use of the secondary electron signal produced in scanning electron (SEMs) or helium ion microscopes (HeIMs) to image the changes in electrical potential caused by the dopant atoms. In this article, the contrast mechanisms and resolution limits of secondary electron dopant contrast are explored. It is shown that the resolution of the technique is dependent on the extent of electrical potential present at a junction and that the resolution of dopant contrast can be improved in the HeIM after an in-situ plasma cleaning routine, which causes an oxide to form on the surface altering the contrast mechanism from electrical potential to material contrast.

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