W.S. Bont

Theory and practice of centrifugal elutriation (Ce): factors influencing the separation of human blood cells

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.

Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique

A modified centrifugal elutriation technique is described for the isolation of large numbers of lymphocytes and monocytes. Elutriation was carried out by lowering the rotor speed at a constant flow rate which was generated by hydrostatic pressure. The flow rate could be kept constant if the separation procedure was performed at high pressure and high systemic resistance. Up to 2.3 X 10(9) mononuclear cells derived from 2000 ml blood were separated in one single experiment in approximately 1 h. The lymphocytes and monocytes were isolated at purities of 98 +/- 1% and 94 +/- 1% respectively. The purity of the lymphocytes was increased to 99.8 +/- 0.1% by a second elutriation run. Additional advantages of the elutriation procedures are that the choice of medium is free, and that relatively large numbers of cells may be separated with high recoveries.

Separation of human lymphocytes and monocytes by velocity sedimentation at unit gravity.

An improved method of separating human peripheral blood lymphocytes and monocytes by velocity sedimentation at unit gravity is described. Cell separation capacity is considerably increased, while the time required for optimal separation is markedly reduced. Simple devices make it possible to layer and fractionate the gradients within 15 min. Overall recovery is 85%. On a small scale, monocytes were isolated greater than 80% pure from 4--20 X 10(7) mononuclear leukocytes. The average yield was 40% of all monocytes recovered after fractionation. In the same sedimentation runs 60% of all recovered lymphocytes were obtained strongly depleted of monocytes (monocyte contamination less than 0.2%). On a large scale 3--6 X 10(8) monocytes with an average purity of 85% were isolated from 3--6 X 10(9) mononuclear leukocytes in 2 subsequent runs. All cells recovered from the gradient were functionally active according to rosette formation, phagocytosis, stimulation by mitogens and in cytotoxicity tests.

Studies on plasma membranes VIII. The effects of sodium deoxycholate and dodecyl sulfate on isolated rat-liver plasma membranes

Abstract 1. 1. Plasma membranes were isolated from rat liver and dissolved in sodium deoxycholate (1%) or dodecyl sulfate (0.4 or 0.8%). Differences in the effects of the detergents on the membranes were observed. 2. 2. Analytical ultracentrifugation showed the presence of heterogeneous membrane material. The aggregation and precipitation of the solubilized membrane material by dialysis appeared to depend on the nature of the detergent used for dissolving the membranes, and the pH and ionic composition of the dialysis fluid. 3. 3. The amount of detergent bound to the dissolved membrane material after treatment with excess detergent solution was determined by three independent techniques based on a similar experimental approach, and a reasonable accordance of results was obtained. The extent of binding appeared to be a function of the detergent concentration and to exhibit an optimum. Dialysis virtually removed the detergents from the membrane material. 4. 4. Density-gradient centrifugation in a three-layer system showed a high degree of dissociation between membrane proteins and phospholipids, both in the presence of detergent and after its prior removal by dialysis. Ultrasonic irradiation also resulted in dissociation. These results were corroborated by equilibrium centrifugation in the analytical ultracentrifuge in a medium of d 1.17, and, in the case of membrane solutions from which deoxycholate had been removed, also by the capacity of the membrane material present in the individual layers of the density gradient after centrifugation to precipitate at pH 5.4. 5. 5. The results are discussed in relation to the problem of lipoprotein subunits in membranes, and the conclusion reached is that the present and other experiments with detergents do not yield evidence for the occurrence of such a structure in the plasma membrane. Criteria for judging whether the original membrane structure is restored in ‘membranes’ reconstructed from their constituting components are presented on account of certain properties of membrane precipitates obtained from deoxycholate solution at pH 5.4.

On the subunits of α-crystallin

α-Crystallin was isolated from total water-soluble lens extract by preparative zone electrophoresis on starch or Pevikon C870 blocks and purified by density gradient centrifugation and Sephadex chromatography. These preparations were treated with urea or sodium dodecyl sulphate and submitted to electrophoresis on polyacrylamide gels containing either urea or sodium dodecyl sulphate. Whereas in 7 M urea a large number of zones was detected, only three bands were observed in 1% sodium dodecyl sulphate. On the other hand, the sedimentation coefficient had the same value in both media. We re-investigated the N-terminal amino acid content in the starch block preparations and compared the result with that obtained from the preparations isolated according to our new procedure. The concentration of dinitrophenyl-glutamic acid was lower in the purified samples. Urea-treated samples had the same concentration of N-terminal glutamic acid whereas the concentration of the “trace” end-groups did not change. Sedimentation-diffusion equilibrium in the analytical ultracentrifuge revealed a slight heterogeneity in the purified samples. Electron micrographs of electrophoretic and of further purified samples were very similar. At neutral or slightly alkaline pH they showed almost uniform spherical aggregates in which a substructure was observed. At acid pH, coiled filaments rather than small globules could be demonstrated.

A recording system for the analytical ultracentrifuge equipped with absorption optics

Abstract A double-beam scanning system is described that can be used in combination with the absorption optics of the analytical ultracentrifuge. A photomultiplier is mounted behind a slit that scans the image of an ultracentrifugal cell. A plot of the light intensity, as a function of the radial distance in a single sector cell, is obtained by appropriate electronic circuits that transform the light signals via the photomultiplier. Either the transmission or the optical density of the solution is plotted; both quantities, their derivatives, or their integrals can be quantitatively recorded. The possibilities of the absorption optics of the analytical ultracentrifuge, when coupled to this automatic scanning device, are illustrated by examples of the following experiments: 1. 1. Sedimentation velocity (polyribosomes). 2. 2. Sedimentation-diffusion equilibrium (subunits of lens protein). 3. 3. Vinograd-band centrifugation (calf-thymus DNA). 4. 4. Density gradient centrifugation with CsCl (mitochondrial DNA). A brief description of the apparatus is given.

An improved method for the separation of cells by sedimentation at unit gravity.

Abstract An apparatus is described for the separation of cells by sedimentation velocity at lg. The viscosity of the sample was raised by the addition of polyethylene oxide and subsequently the sample was layered on top of a density gradient via a sieve. With the aid of this procedure the various ploidy classes of rat-liver cells were enriched and murine leukemia cells were separated according to the phases of their life cycle.

An apparatus for preparative polyacrylamide electrophoresis: The isolation of a ribonuclease inhibitor

Abstract A postmicrosomal supernatant from rat liver, containing an inhibitor of ribonuclease, was prepared. The inhibitor was concentrated in a “pH 5” supernatant. The partially purified inhibitor was fractionated on a column of CM-cellulose. The fraction containing the inhibitor was further purified by electrophoresis on polyacrylamide gel. The apparatus for performing the preparative electrophoresis with polyacrylamide gel is described.

Elongation factor 2 as the target of the reaction product between sodium selenite and glutathione (GSSeSG) in the inhibiting of amino acid incorporation in vitro

The product of the reaction between sodium selenite and glutathione, designated as selenodiglutathione (GSSeSG), nearly completely inhibits amino acid incorporation from [14C]leucyl-tRNA by free polyribosomes isolated from rat liver. The mechanism of this inhibition was studied on the basis of the following three findings. Glutathione decomposes GSSeSG to harmless products; GSSeSG acts instantaneously on some component of the complete incubation system during preparation of the incubation vessels (at 0 degrees C); once GSSeSG has reacted its inhibitory effect cannot be reversed by glutathione. Accordingly, the effect of GSSeSG on the various steps of the amino acid incorporation process was studied by varying the sequence of additions of the reaction components, GSSeSG and GSH. The results of these and other experiments showed elongation factor 2 to be target of GSSeSG. The GSSeSG-B blocked factor could be regenerated by reduction with glutathione reductase and NADPH.

Inhibition of amino acid incorporation in a cell-free system and inhibition of protein synthesis in cultured cells by reaction products of selenite and thiols

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 microM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 microM to over 7 microM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 microM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.