Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation

All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymerase chain reaction (PCR) amplification of a 16S rRNA gene fragment, followed by hybridization of the amplified fragments to a set of specific probes to assess the phylogenetic diversity of our samples. Control parameters, such as the relationship between amount of DNA template and amount of PCR product and the influence of competing DNA on PCR amplification, were first examined. Comparison between extraction methods showed that less DNA was extracted when cells were first separated from the soil matrix (0.4 µg g⁻¹ dry weight soil versus 38–93 µg g⁻¹ obtained by in situ lysis methods). However, with the exception of the γ-subclass of Proteobacteria, there was no significant difference in the spectrum of diversity resulting from the two extraction strategies.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

Summary A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

The morphology of erythroid cells separated by density gradient centrifugation through ficoll

Summary The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.Supported by grants from the National Research Council of CanadaWe thank Dr. G. Setterfield for assistance with the EM data and we are grateful to the N.R.C. for use of centrifuges and the zonal rotor     

Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation

We investigated relationship between the maturity and density of muscle cells and developed a rapid isolation method to acquire stem cells from skeletal muscle. Mononuclear cells were isolated from the lower hind-limb muscles of 7-d-old male Sprague–Dawley rats and separated by Percoll density gradient centrifugation. After centrifugation, the cells were layered in the interfaces between each Percoll density layer. Flow cytometry was used to investigate the Sca-1, Pax7, CD34, CD45, M-cadherin, and myosin expression of the cells in each density layer. We found that CD45-positive cells were not present in freshly isolated muscle cells. CD34-, Pax7-positive cells were mainly observed at the interface between the 15% and 25% Percoll layers and had a density of 1.0235–1.0355 g/ml. Cells positive for M-cadherin were at the 25–35% Percoll density interface and had a density of 1.0355–1.0492 g/ml. We conclude that because there appears to be a correlation between maturity and density, muscle-derived stem cells may be isolated successfully from the 15–25% Percoll interface.     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation

Summary Two methods for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23–1.30), which lack cytochrome c oxidase activity.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. Yasuda     

Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since “intermediate DNA” following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease.When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 10⁶) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons.If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, “intermediate DNA” with similar properties is found between the light-heavy and heavy-heavy peaks.     

Preparative density gradient centrifugation of RNA and DNA in cesium sulfateurea mixture

A new procedure is described for a large scale separation and purification of unfixed DNA and RNA from a mixture of partially extracted nucleic acids and lysates of subcellular fractions by centrifugation to equilibrium in cesium sulfate-urea mixture. Optimum conditions are described for the separation and quantative recovery of both RNA and DNA in a pure form. The procedure allows determination of peak buoyant densities of 4–5s RNA, 7–11s mRNA and total cytoplasmic RNA. The procedure also allows fractionation of small molecular weight classes of cytoplasmic RNAs from the 18s and 28s rRNAs.     

Selective autophagy of clofibrate-induced rat liver peroxisomes. Cytochemistry and immunocytochemistry on tissue specimens and on fractions obtained by Nycodenz density gradient centrifugation.

Following short treatments with peroxisomal proliferators rodent liver undergoes a significant increase in the peroxisomal population, accompanied by specific induction of some peroxisomal enzymes; both phenomena are reversible and in a few days after drug withdrawal the control parameters are recovered. The involvement of lysosomal system in removal of proliferated peroxisomes has been widely suggested, and the autophagic phenomenon was mainly investigated in experimental conditions in which the administration of lysosomotropic drugs or, more generally, of digestive process inhibitors caused an accumulation of autophagic vacuoles. In the present research the removal of clofibrate-induced rat liver peroxisomes was investigated under physiological conditions, i.e. in the absence of drugs interfering with the autophagic process. In a previous paper the lysosomal involvement in peroxisomal removal was suggested on the basis both of biochemical and cytochemical-immunocytochemical data. In the present paper the autophagic vacuoles and autolysosomes involved in the digestion of excess peroxisomes are more extensively described, mainly by means of colloidal gold immunocytochemistry, carried out also on density gradient subfractions.     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation.

Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.