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 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 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 marine seston and density determination of marine diatoms by density gradient centrifugation

An attempt has been made to separate constituents of marineseston samples: inorganic material, detritus and the algal species,by density gradient centrifugation, without affecting the physiologicalstate of the algae. A relatively inert gradient material, consistingof Percoll, salt and sucrose, was composed. Since the densitiesof detritus and algae as well as those of different algal speciesoften overlapped, only 10 of the 100 samples processed in thecourse of the year showed a reasonable separation. However,an enrichment with respect to one or more species was oftenachieved. Densities of eleven species of marine diatoms andof one dinoflagellate have been determined at different timesof the year. For eight diatom species and for the dinoflagellatethe following specific density ranges were established: Bidduiphiaaurita: 1.18–1.23 g cm^–3, Biddulphia sinensis: 1.03–1.08g cm^–3, Cerataulina bergonii: 1.03–1.06 g cm^–3,Ditylum brightwellii: 1.07–1.13 g cm^–3, Rhizosoleniadelicatula: 1.04–1.09 g cm^–3, Skeletonema costatum:1.12–1.17 g cm^–3, Streptotheca thamensis: 1.04–1.10g cm^–3 , Thalassiosira rotula: 1.05–1.10 g cm^–3,Peridinium sp.: 1.08–1.12 g cm^–3. No seasonal variationin density was demonstrated. Gradients of different compositiondid not influence density measurements.     

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.     

Separation of oyster hemocytes by density gradient centrifugation and identification of their surface receptors

Glutaraldehyde-fixed hemocytes of Crassostrea virginica were subjected to differential centrifugation on a 5, 10, 15, and 25% discontinous sucrose gradient. Five subpopulations of cells were separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation 2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation 4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes and aggregates of small cells. By using several plant and animal lectins, it was ascertained that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the albumin glands of Helix pomatia and Cepaea nemoralis while those of subpopulation 2 were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying the Con A-peroxidase cytochemical technique, it was determined that approximately 20% of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18% of the large cells comprising subpopulation 5 possess such sites. These results suggest that the subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may be further subdivided by differences in specific surface binding sites.     

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.     

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.     

Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.