Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

A new method designed for the specific isolation and characterization of ligand-receptor complexes using a heterobifunctional crosslinking agent and immunoprecipitation is described. The complexes are first covalently crosslinked by photoactivation of the crosslinking agent. After lysis of the cells, the crosslinked complexes are immunoprecipitated using an antiserum directed against the crosslinking agent. With this method, ligand-receptor complexes formed in only minute amounts become available for further investigation. By using this anticrosslinker antiserum, different receptor systems can be investigated without raising new receptor- or ligand-specific antibodies for each system. As a test system, a radioiodinated lectin was used as ligand molecule and erythrocyte membranes acted as receptor carriers.     

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.     

Removal of contaminating proteases from Bacillus thuringiensis parasporal crystals by density gradient centrifugation in NaBr

Summary Purification of the Bacillus thuringiensis protein crystal on NaBr density gradients confers a significant technical advantage in that the crystal-associated proteases are thereby removed. The use of protease-free crystals allows reliable determination of native crystal parameters.     

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.     

Separation of small ciliate protozoa from bacteria by sucrose gradient centrifugation.

Small ciliate protozoa of the genus Uronema were separated from labeled bacteria by means of a discontinuous sucrose gradient. More than 98% of the radioactivity associated with the bacteria was found in the upper portion of the gradient, whereas the remaining portion of the gradient contained the ciliates.     

Separation of planarian neoblasts based on density gradient centrifugation

For highly purified preparations of neoblasts, density gradient centrifugation in Percoll solutions (Pertoft et al., 1978) was applied to cell suspensions obtained by disintegrating Dugesia polychroa (Schmidt) in culture medium contained in a Dounce homogenizer (tolerance: 50 µm; one animal 12 mm in length per ml). To reduce the high viscosity caused by mucus, 0.00063% (w/v) of dithiothreitol was added during disintegration and purification. Based on previous experiments (Schürmann & Peter, 1988), five media were compared.For prepurification, four washing steps (differential centrifugations at 500 × g for 5 min each) were followed by subsequent filtration through a series of nylon gauzes (40, 30, 20 and 15 µm mesh size) and a final washing step. The resulting cell suspensions were then fractionated by isopycnic centrifugation (500 × g, 45 min) in one continuous (1.018–1.121 g ml^–1) or one of seven different discontinuous Percoll gradients (Schürmann, 1993). The best yield and highest purity of neoblasts in one fraction was obtained with a four step gradient (1.03–1.09 g ml^–1): the neoblasts (purity: 91%) were concentrated in one sharp band at the boundary between the densities 1.05 and 1.07 g ml^–1. The spherical cells (diameter from 10 to 13 µm in vivo) stained as typical neoblasts (Pedersen, 1959).Primary cultures were obtained with all media. The medium developed by Teshirogi and Tohya (1988) and its isotonic modification (Schürmann, 1993) proved best, resulting in 86% of viable cells without signs of differentiation after 17 days of culture at 18 °C, with still 46% being left after 31 days. Earlier reports state that isolated neoblasts only survive for 4 days (Betchaku, 1967) and total planarian cell suspensions only 2–3 weeks (Teshirogi & Tohya, 1988).     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

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; )     

Remarks on the differentiation of lysosomes from cultured human fibroblasts by silica gradient centrifugation

Two bands of lysosomal marker enzyme activity from cultured skin fibroblasts can be obtained by silica gradient centrifugation (Rome, L H, Garvin, L H, Allietta, M M & Neufeld, E F, Cell 17 (1979) 143) [3]. It is shown that the distribution of lysosomes into the bands in the upper and lower part of the density gradient depends on the starting density and the shape of the self-forming gradient. A plot of marker enzyme activity vs density reveals that the two bands stem from a lysosomal population with unimodal density distribution.     

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.