Differences In Buoyant Density Research Articles

Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy.

Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index ( n ≈ 1.35) compared with the LD EVs ( n ≈ 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.

Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ∼700-900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000-1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ∼1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ∼170-μm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800-1,700 nm).

Probing Carbon Nanotube–Surfactant Interactions with Two-Dimensional DOSY NMR

Two-dimensional diffusion ordered spectroscopy (2D DOSY) NMR was used to probe the micellar structure of sodium dodecyl sulfate (SDS) and sodium cholate (SC) in aqueous solutions with and without semiconducting and metallic single-walled carbon nanotubes (SWCNTs). The solutions contain SDS and SC at weight ratios of 1:4 and 3:2, the ratios commonly used to isolate semiconducting and metallic SWCNTs through density gradient ultracentrifugation (DGU). These results show that the coverage of surfactant on the semiconducting and metallic SWCNTs is nearly identical in the 1:4 surfactant mixture, and a lower degree of bundling is responsible for the greater buoyancy of semiconducting SWCNTs. In the 3:2 surfactant mixture, the metallic SWCNTs are only encapsulated in SC while the semiconducting SWCNTs remain encapsulated in a poorly packed two-surfactant micelle, leading to a large buoyant density difference between the electronic species. This work provides insight into future directions to increase the purity of semiconducting and metallic SWCNTs sorted through DGU and demonstrates the utility of 2D DOSY NMR in probing SWCNT-surfactant complexes.

Mosquito Mass Rearing Technology: A Cold-Water Vortex Device for Continuous Unattended Separation of Anopheles arabiensis Pupae from Larvae

In mass rearing of anopheline mosquitoes, pupae are usually separated from larvae on a daily basis to prevent unwanted adult emergence from trays. Depending on the device and species, 2 physical characteristics have most often been used for separation: buoyant density and size. In this report, we describe a system for continuous separation of Anopheles arabiensis larvae from pupae based on the natural difference in buoyant density and behavior between the 2 stages. We determined that temperatures 4-15 degrees C caused neither mortality nor reduction in likelihood of pupation or emergence. Separation improved as temperatures decreased down to 4 degrees C. We devised and demonstrated a 15 degrees C water vortex separator that we anticipate can process approximately 1 million larvae and pupae per hour with a < 0.3% pupal contamination rate and which operates unattended.

Method for the Isolation of &lt;em&gt;Francisella tularensis&lt;/em&gt; Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (<10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of &lt;em&gt;Francisella tularensis&lt;/em&gt; Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (<10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

&lt;em&gt;In-vivo&lt;/em&gt; Centrifugation of &lt;em&gt;Drosophila&lt;/em&gt; Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle.Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

&lt;em&gt;In-vivo&lt;/em&gt; Centrifugation of &lt;em&gt;Drosophila&lt;/em&gt; Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

Separation and Characterization of Double-Wall Carbon Nanotube Subpopulations

Surfactant-encapsulated double-wall carbon nanotubes (DWCNTs) synthesized by the high-pressure carbon monoxide decomposition (HiPco) process were separated by length and electronic characteristics using density gradient ultracentrifugation (DGU). To ensure our study focuses only on the behavior of DWCNTs, dispersed DWCNTs were first isolated following the method of Green et al. [Green, A. A.; Hersam, M. C. Nat. Nanotechnol. 2008, 264, 1], utilizing the differences in buoyant density of DWCNTs from that of impurity single-wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes contained in the parent soot. By increasing the density difference between the nanotubes and the density gradient medium, we exploited the length-dependent translation of the nanotubes in response to applied centrifugation to isolate narrow length distribution DWCNT fractions. The length-dependent intrinsic optical response of DWCNTs is consistent compared with the previously reported values for SWCNTs. The controlled addition ...

Separation of PHA-accumulating cells in activated sludge based on differences in buoyant density

Separation of PHA-accumulating cells in activated sludge based on differences in buoyant density

Sorting Carbon Nanotubes for Electronics

Because of their unique structure and composition, single-wall carbon nanotubes (SWNTs) are at the interface between molecules and crystalline solids. They also present properties that are ideal for making lightweight, inexpensive, and flexible electronics. The raw material is composed of a heterogeneous mixture of SWNTs that differ in helicity and diameter and, therefore, requires purification and separation. In a series of groundbreaking experiments, a robust process serving this purpose was developed based on SWNTs encapsulated in surfactants and water. Ultracentrifugation in a density gradient combined with surfactant mixtures provided buoyant density differences, enabling enrichment for both diameter and electronic properties. A new paper in this issue explores further the process through the hydrodynamic properties of SWNT-surfactant complexes. The study reveals that we have just begun to uncover the dynamics and properties of nanotube-surfactant interactions and highlights the potential that could be gained from a better understanding of their chemistry. The time scale of integration of carbon nanotubes into electronics applications remains unclear, but the recent developments in sorting out SWNTs paves the way for improving on the properties of network-based SWNTs.

Origins and consequences of mitochondrial decline in nucleated erythrocytes

Cellular aging in nucleated erythrocytes from lower vertebrates is accompanied by losses in mitochondria but it remains unclear (i) how these losses accrue (ii) if these changes alter energetics and (iii) whether such changes increase the propensity for apoptosis. We addressed these questions using trout erythrocytes that were separated into age classes using inherent differences in buoyant density. The oldest cells showed a profound decline in mtDNA transcripts, due to reductions in both transcription (90% decline in total RNA) and mtDNA copy number (35%). No alterations in the ratio of 16S rRNA to COX I mRNA were detected, nor was there an accumulation of unprocessed mtDNA transcripts. While older cells had reduced basal respiration, there were no changes in mitochondrial enzymes stoichiometries, tissue ATP levels or dinitrophenol-induced (maximal) respiration rates. Apoptosis could not be induced in either whole blood, young or old erythrocytes by pro-oxidants, mitochondrial inhibitors or staurosporine. In contrast, cyclosporin A (CsA) caused caspase 3 activation, DNA laddering and LDH leakage, but only in young cells. Both CsA and a combination of azide, oligomycin and dinitrophenol cause mitochondrial depolarization and caspase 9 activation, but only CsA induced caspase 3 and apoptosis. Caspase inhibitor studies support the conclusion that mitochondrial changes may accompany CsA-induced cell death, but are not essential in its progression. While pifithrin failed to induce cell death, it enhanced the effects of CsA, implicating a role for p53. Collectively, these studies suggest that the mitochondrial changes with aging do not compromise cellular function, although trout erythrocytes can initiate apoptosis by non-mitochondrial pathways.

Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase.

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase-coupled transition of E. coli phenotype is discontinuous.

A ComGA-dependent checkpoint limits growth during the escape from competence.

In Bacillus subtilis, competence for transformation develops in 5-10% of the cells in a stationary phase culture. These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence. To better understand the basis of this lag, we have characterized competent cultures microscopically. To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker. Only 5-10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event. To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK-gfp. Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings. Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h. In contrast, the majority of non-competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ-ring formation. The late competence protein ComGA is required for the competence-related block in chromosome replication and cell division. In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ-ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance. It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation. A mutation in the ATP-binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division. The buoyant density difference between competent and non-competent cells depends on the competence-specific growth arrest.

The Sugar Content and Density of Living and Dead Microsporidian (Protozoa: Microspora) Spores

Microsporidian (Microspora) spores were subjected to several kinds of stress: ultraviolet light (Nosema algerae); chilling in ice water (Edhazardia aedis); freezing and thawing, with or without glycerol as a cryoprotectant (Nosema disstriae, Thelohaniasp.); long-term cold storage (Vairimorphaspp.,Endoreticulatussp.,Thelohaniasp., andMicrosporidiumsp.); and extended periods at room temperature (Microsporidiumsp. andVairimorpha lymantriae).N. algeraespores were inactivated by incubation in 0.05mNaCl or 24 hr at 23°C. Viability of the spores was assessed either by infectivity to a susceptible host or byin vitrogermination. Carbohydrates were extracted from the spores for measurement of the total sugars (anthrone reactive) and reducing sugars (Nelson's test), and quantities and ratios of the two classes of sugars were compared. Buoyant density differences associated with sugar concentration and viability changes were estimated by Ludox density gradient centrifugation. Reducing sugar levels increased after germicidal ultraviolet radiation, chilling, or freezing, concurrent with loss of infectivity or capacity for germination. The smaller reducing sugar molecules apparently diffused slowly from the inviable spores, causing a gradual reduction in the total concentration of sugars. Many samples of spores held for long periods in cold storage were almost devoid of sugar. These changes were not seen inN. algeraespores that were temporarily inactivated. In all cases, spores that were severely depleted in carbohydrates had a significantly lower buoyant density. The bands of heavy spores had a mean density ± SE of 1.197 ± 0.010 g/ml and the mean density of the light, sugar-depleted spores was 1.137 ± 0.024 g/ml. These results suggest that, where no other means are possible, sugar concentrations or spore density might be used to assess the viability of spores.

Isolation and culture of somatotrophs from the pituitary of the rainbow trout: immunological and physiological characterization.

The aim of the present paper was to obtain somatotroph- and gonadotroph-enriched populations from collagenase dispersed pituitaries of male rainbow trout. Inasmuch as the percentage of immunoreactive gonadotrophs and somatotrophs present in pituitaries was higher at spermiation than at the beginning of spermatogenesis, we tried such a cell separation with fish at this stage of spermatogenesis. Cells were fractionated using their differences in buoyant density with centrifugation in Percoll solutions. The use of Percoll linear gradients (1.110 to 1.027 g/ml) showed that somatotroph cells have a density of between 1.102 and 1.064 g/ml whereas gonadotrophs are spread over the range of the gradient. It was thus possible, by using linear or discontinuous Percoll gradients, to obtain 95 to 67% (mean 80%) enriched somatotropic cell fractions while no enriched gonadotropic cell fractions were collected. The fractionated cells kept their ability to be cultured and to be responsive to specific secretagogues. Somatostatine induced a 80 to 85% decrease in growth hormone release per somatotroph in the initial cell suspension as well as in the different cell fractions. On the other hand, the basal growth hormone release per cell was lower in the fractions containing cells with a density lower than 1.062 g/ml. Inversely, the gonadotrophs have a basal release per cell independent of their density, and this is also available for their responsiveness to salmon gonadotropin-releasing hormone.

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

The use of targetted liposomes to isolate cells bearing immunoglobulin receptors

A new method of mouse B cell isolation has been developed. It is based on the differences in buoyant density of free lymphocytes and cells bound within liposome-cellular complexes (LCC). LCC arise during the interaction of B cells bearing surface immunoglobulins (Ig) with liposomes loaded with affinity purified rabbit antibodies to mouse IgM/IgG. Advantage was taken of the fact that LCC can be selectively removed from other cell populations by differential flotation in Ficoll gradients. Because of their lower density the LCC did not sediment under conditions where free lymphocytes and complexes of the liposomes with dead or damaged lymphocytes did sediment. The method was highly specific and the recovery of B cells was about 50%. B cells isolated with the use of targetted liposomes retained viability and functional activity. Theoretically, liposomes loaded with antibodies to any cell surface marker could be used for the isolation of cells bearing such markers.

Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5′ and 3′ nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour.

Density distributions of human squamous carcinoma cells: Influence of growth conditions, proliferative status and DNA content

Human squamous carcinoma cells (A431, CaSki) were grown in vitro as multicellular spheroids or as exponential and plateau-phase monolayer cultures. Single-cell suspensions were obtained by disaggregating the spheroids and monolayer cultures with trypsin-EDTA. Their buoyant density distributions were then compared by centrifuging the suspensions for 30 min at about 800gave and 4 degrees C in linear, continuous Percoll gradients. We found differences in buoyant density related to the growth condition (i.e. cells grown as spheroids or plateau-phase cultures were more dense than those from exponential cultures) and to the cell-line (i.e. CaSki cells were more dense than the A431 cells). Cells isolated from the inner layers of the spheroids appeared to be less dense than those from the outer layers. In addition, quiescent cells separated from the A431 spheroids by centrifugal elutriation, a technique based on cell size, were more dispersed in buoyant density than the corresponding proliferating cells recovered from the same spheroids. Flow cytometric analysis with mithramycin was performed to measure the DNA content of the cells. We found that the CaSki cells contained relatively more DNA than the A431 cells whereas, for the same cell line, growth as monolayer cultures or as multicellular spheroids did not influence their relative DNA content. We conclude that the growth conditions, the proliferative status of the cells and possibly their relative DNA content may influence the density distribution of these cells.