Separation of ram spermatids by sedimentation at unit gravity

Suspensions of 1 × 10⁸ ram testis cells were prepared with trypsin and separated by velocity sedimentation at unit gravity in a non-linear Ficoll gradient. An improvement was made in the technique of cell suspension preparation to increase the viability of heat-sensitive germ cells. Six bands of cells numbered from I–VI were characterized by their sedimentation velocity and modal cell volume. The distribution of various classes of germ cells in these bands, and especially spermatids at different maturation stages, was determined using histological techniques and confirmed by kinetic profiles and autoradiographic analyses of ³H-thymidine incorporation. A homogenous population of round spermatids (93–96%) was obtained in the high sedimentation velocity part of band IV. Although elongated spermatid separation does not rigorously follow the maturation stage, it gives populations which can be used for the investigation of biochemical changes during spermiogenesis. Taking into account the viability and the ultrastructure of germ cells after separation, it was shown that the use of Ficoll as a gradient material instead of bovine serum albumin causes cellular damage. We successfully applied the technique of velocity sedimentation to testis cell separation in the bull, boar, billy-goat and stallion.     

Analysis of rat adrenal cells in primary culture separated by velocity sedimentation

Summary Cell separation was used to follow the fate of the cortical cells of the adrenal gland in primary culture, and to assess some of the changes that occur as cells adapt to culture conditions. Primary cultures of rat adrenal gland were dissociated with trypsin and separated by velocity sedimentation at unit gravity. After two days in culture, cells showed a reproducible sedimentation profile consisting of two classes of cells with mean sedimentation rates of 5.8 and 2.1 mm/h, and a third sedimentation peak consisting mainly of nuclei at 0.5mm/h. All populations continued to incorporate ³H-thymidine in relatively constant proportion throughout the culture period, but the relative number of cells in the 2.1 mm/h peak increased two-fold in the last few days of primary culture. Cells labelled in primary culture, but separated after an additional 5 days in secondary culture had lost proportionately more labelled cells from the 5.8 mm peak. The results suggest that cells of the 2.1 mm peak survive longer in culture in a post-replicative condition.Work reported in this paper was performed while the author was a Research Fellow of the National Cancer Institute of Canada in the laboratory of Dr. N. Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada     

Unit gravity sedimentation separation of cells comprising the caput epididymidis of the rat

Summary Cells from the rat caput epididymidis were separated by unit gravity sedimentation. Purest 12-ml fractions contained 88–95% basal cells or 64–76% principal cells. Ultrastructure of separated cells was similar to that of cells in intact tissue. Viability of separated cells was excellent as determined by dye exclusion tests and cellular ATP content. By combining fractions pools containing 4.0±0.9 × 10⁶ cells (86±8% basal cells) and 1.4±0.4 × 10⁶ cells (56±7% principal cells) were obtained. Thus, studies on the function of basal and principal cells from the rat caput epididymidis should be possible.This research was supported by NIH Grant HD-07244 and was authorized for publication as Paper No. 5231 in the Journal Series of the Pennsylvania Agricultural Experiment Station. We thank Mrs. J. Eastman for expert technical assistance     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Separation of primitive and definitive erythroid cells of the chick embryo

The primitive and definitive erythroid cells of the chick embryo are separated preparatively by means of velocity sedimentation at unit gravity in BSA gradients. Analyses of the hemoglobins contained by the fractionated cells show a segregation of different hemoglobins between the primitive and definitive cells. Studies of the incorporation of [³H]leucine show that the fractionated cells are normal with respect to their protein synthetic activities and that their relative rates of incorporation are markedly different.     

Separation of Marine Bacteria according to Buoyant Density by Use of the Density-Dependent Cell Sorting Method

The purpose of this study was to test whether some phylogenetic groups of natural marine bacteria have unique buoyant densities that allow them to be separated by the density-dependent cell sorting (DDCS) method. We first concentrated a natural bacterial assemblage to collect sufficient numbers of cells. They were separated into three fractions by DDCS, and the community structure in each was clarified by fluorescence in situ hybridization. The cells of Archaea tended to appear in the high-density fraction, whereas those of Cytophaga-Flavobacterium-Bacteroides were in the low-density fraction. We also calculated the sedimentation velocities of three typical marine bacteria (low density, middle density, and high density) using their buoyant density. The sedimentation velocities were approximately 10, 20, and 30 μm h⁻¹; these velocities have ecological implications when the heterogeneity of bacteria is considered at a microscale. To our knowledge, this is the first report on the buoyant density of natural marine bacteria.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

SEPARATION OF NUCLEI REPRESENTING DIFFERENT PHASES OF THE GROWTH CYCLE FROM UNSYNCHRONIZED MAMMALIAN CELL CULTURES

Nuclei have been isolated from unsynchronized cultures of Chinese hamster fibroblasts after varying intervals of growth following the incorporation of thymidine ^-3H for 20 min. These nuclei were fractionated by unit gravity sedimentation in a stabilizing density gradient of sucrose, and fractions were analyzed for the concentration of nuclei, DNA, and radioactivity. A more rapidly sedimenting population of nuclei in the G₂ phase of the cell cycle was separated from a group of nuclei in the G₁ phase, and nuclei in progressive stages of DNA synthesis (S phase) were distributed between these two regions. The fractionation of intact cells by sedimentation according to their position in the cell cycle was found to be less satisfactory than the corresponding separation of nuclei. This probably results from the continuous accumulation of mass within individual cells throughout the entire cell cycle, whereas most of the mass of a nucleus is replicated during a relatively narrow interval of the total cell cycle.     

Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of ³H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.     

Zonal unit-gravity elutriation

A new and simple technique, zonal unit-gravity elutriation, has been devised for separating very large cells, multicellular complexes, or small organisms from suspensions consisting mainly of small cells. The separation vessel is a conical chamber with an entrance at the lower, narrower part of the cone and an exit at the upper, wider part of the cone via a dome-shaped lid. A baffle at the entrance prevents turbulence from incoming fluid. Chambers of differing widths and wall slopes are chosen depending on the sedimentation rate of the particles to be separated. A small volume of the cell suspension is placed in the chamber on the bench in a cold-room. Medium stabilized by a shallow density gradient is pumped into the base of the chamber and ascends, creating a decreasing velocity gradient. Cells sediment at unit-gravity against this ascending counterstream, and are separated into bands according to sedimentation velocity. By adjusting the flow rate of the medium, different sizes of cells can be separated. Tumor cells can be enriched, and larger blast cells can be separated from small cells in lymphoid cell suspensions. The procedure produces complete separation of thymic nurse cells (epithelial-lymphoid complexes) from free thymocytes in digested thymus suspensions and produces substantial enrichment of thymic rosettes (macrophage-lymphoid complexes). A very favorable situation for applying this technique is the isolation ofTaenia taeniaformis larvae, which can be completely purified from infected liver suspensions, representing a 4×10⁵-fold enrichment of the parasites, with high recovery, in a single 30 min operation.     

Biochemical analysis of meiosis in the male mouse

Spermatogenic cells of the mouse have been separated by gravity sedimentation using a modification of a previously published method. Details are given for the collection of purified samples of specific meiotic stages and for the collection of labelled cell fractions following injection of ³H-thymidine. Suppression of semi-conservative meiotic DNA synthesis, essential to the biochemical analysis of pachytene DNA metabolism, has been achieved by in vivo administration of 1 M hydroxyurea.     

Sedimentation properties of chitosomes fromMucor rouxii

Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm³; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl₂ was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl₂ led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.     

Ontogeny of cellular immunity: size and turnover of rat thymocytes responsive to in vitro stimulation

Velocity sedimentation of thymus cell suspensions prepared from Wistar rats of different ages has been used to separate cells mainly on the basis of size. Large cells, many in division, sediment faster than the major population of small thymocytes and can be separated from them on this basis. In vitro responsiveness to mitogens has been found to be associated with the minor populations of larger cells, particularly the medium-large cells (230–270 μm³). The mitogen-responsive populations also contain cells which show a high autonomous DNA synthesis.The size distribution of thymus cells in neonatal animals is more varied than in adults and there is frequently a higher proportion of large cells. The mitogen-responsive cells were again found among the fast sedimenting large cells and the highest responses were seen with the very largest cells (approximately 450 μm³).The cells in both adult and neonatal animals which have the capacity to respond to mitogens in vitro are probably larger in size than the normal peripheral recirculating thoracic duct cells. The major population of normal small thymocytes showed a much lower spontaneous uptake of ³H-thymidine and did not respond to mitogens.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Biochemical determination of tubulin-microtubule equilibrium in cultured cells.

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH₃ cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34–41% of tissue culture cell tubulin. Colchicine (10^?6 to 10^?5m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Isolation of plasma membranes from Xenopus embryos

Summary Plasma membranes were isolated in high yield from Xenopus gastrulae by repeated sedimentation in discontinuous sucrose gradients. Most of the yolk was separated by lowspeed sedimentation before centrifugation on the discontinuous sucrose gradients. The isolation of plasma membranes was followed by covalent labelling of the surface of dissociated gastrula cells with diazoniobenzene sulphonate, by electron microscopy and the distribution of enzymatic markers. The isolated plasma membranes have a low neural inducing activity as compared to other cell constituents.