Separation and subfractionation of small numbers of cells (∼106) by countercurrent distribution in dextran-poly(ethylene glycol) aqueous-phase systems

Separation and subfractionation of cells on the basis of subtle differences in surface properties by partitioning in dextran-poly(ethylene glycol) aqueous phase systems is an established method. We report here that the incorporation of fetal bovine serum into such systems permits countercurrent distribution of small quantities of cells (∼10⁶). In the absence of serum such small quantities of cells are lost (probably by adherence) and cannot be recovered after countercurrent distribution.     

Separation of human rosette- and nonrosette-forming lymphoid cells by countercurrent distribution in an aqueous two-phase system.

Mixtures of aqueous solutions of dextran and of poly(ethylene glycol) form immiscible two-phase systems suitable for the separation (by partition) of cells based on subtle differences in membrane surface properties. Lymphoid cells from human tonsils were subjected to countercurrent distribution in such a system. Analysis of cells from different parts of the countercurrent extraction train by the sheep red blood cell rosette test indicates a separation of rosette-forming and nonrosette-forming lymphocytes with the former having the higher partition coefficient. Since a major determinant of cell partition in these phases is a membrane-charge associated property it appears likely that the rosette-forming cells have a higher surface charge than the nonrosette-forming cells.     

Blood clam (Anadara inflata) red cells. partition in aqueoues two-polymer phase systems

Anadara inflata is a clam which has red blood cells in its hemolymph. Furthermore, the nucleated red blood cells contain two structurally distinct hemoglobins. Clam red cells were subjected to partition in aqueous dextran-polyethylene glycol two-phase systems with the following results:

1.

1. Clam red cells are the largest cells (about 20 μm in diameter) so far studied in two-polymer phases. It is shown that not only can such cells be partitioned in dextran-polyethylene glycol phase systems, but that countercurrent distribution resolves the clam red cell population into more and less metabolically active cells. The distribution of these cells in relation to the whole population is similar to that of young and old red cells from mammals.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems. I. Characterization of subpopulations of cells.

Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems.

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharide is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.     

Cell separations and subfractionations by countercurrent distribution in two-polymer aqueous phase systems depend on non-equilibrium conditions

Certain kinetic aspects of the partitioning behaviour of erythrocytes in dextran-poly(ethylene glycol) aqueous phase systems have been examined, and their implications for cell partitioning have been considered. It is concluded that the diverse and sometimes unique fractionations attained by use of multiple extractions (e.g., countercurrent distribution) of cells in two-polymer aqueous-phase systems depend on non-equilibrium conditions.     

Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharides is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.     

Countercurrent distribution and multiple sedimentation of Chlorella pyrenoidosa during its life cycle

Synchronously and normally grown Chlorella pyrenoidosa cell populations were analysed by countercurrent distribution in aqueous two-polymer phase systems and by a multiple sedimentatation technique. Partition of cells in aqueous phases reflects the surface properties of cells (primarily surface charge) and multiple sedimentation reflects the cells' size-density parameters. It was found that:

1. 1. Synchronized cells that have just divided have the lowest partition of any in the population. Surface charge (as reflected by partition) increases with time after cell division. Cells have the highest partition just prior to division.

2. 2. Synchronized cells that have just divided are the smallest of any in the population. Since size and sedimentation rate increase with time after cell division multiple sedimentation permits the separation of cells of different ages.

3. 3. Both countercurrent distribution and multiple sedimentation studies reveal considerable heterogeneity of synchronized Chlorella populations. The increase in both surface charge and size with cell age does not appear to proceed in a continuous fashion. Rather, it seems to go in a stepwise manner.

4. 4. Non-synchronized cells examined by either countercurrent distribution or multiple sedimentation show two distinct sub-populations. One of these corresponds to the youngest, just divided cells; and the other to cells just prior to cell division. It is suggested that a lag time just prior to cell division and just after cell division explains these results.

5. 5. Countercurrent distribution in two-polymer phases and multiple sedimentation at unit gravity in a suspension medium best suited for the cell under investigation seem to be methods of choice for tracing cell changes during division, maturation and aging and for sub-fractionating such cell populations.

     

高速逆流色谱技术在生物大分子分离纯化中的应用

高速逆流色谱是一种连续液-液色谱技术,具有无固相载体、样品无需严格预处理等优点。近10年来,在设备结构和溶剂体系等方面进行了大量的研究开发,已推广应用于生物技术、医药、天然产物、环境监测、食品等领域。为适应生物大分子和活性细胞的分离,采用条件温和的双水相体系,研究开发相应的高速逆流色谱设备已成为热点。针对双水相体系的特点,已经开发出了多种具有较高固定相保留率的新型高速逆流色谱设备,通过优化实验条件,成功地进行了多种蛋白质的分离纯化。本对该领域的最新进展进行了综述与评价。     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Membrane surface properties of lymphocytes of normal (DBA/2) and autoimmune (NZB/NZW)F1 mice: effects of L-canavanine and a proposed mechanism for diet-induced autoimmune disease

Partitioning cells in a dextran polyethylene glycol aqueous two-phase system (countercurrent distribution, CCD) is a sensitive method for learning about cell surface membrane properties and for subfractionating cell populations. In this study, we subjected lymphocytes from normal DBA/2 mice and autoimmune F1 New Zealand black/New Zealand white [NZB/NZW)F1) mice to countercurrent distribution and found that T cells partition to the right and B cells partition to the left of the CCD curve. We found no difference between the CCD patterns of normal and autoimmune mice. When the murine lymphocytes were exposed to a cationic dietary amino acid (L-canavanine) in vitro, L-canavanine selectively affected the CCD pattern of autoimmune B cells, reflecting an alteration in surface membrane properties. We separated these lymphocytes with altered surface membrane properties by CCD. Impaired B-cell immune responses associated with L-canavanine were isolated to this lymphocyte fraction. This study provides the first evidence that alterations in the charged surface membrane properties are associated with abnormal (auto) immune response.     

Histochemical and immunohistochemical localization of hexokinase isoenzymes in rat kidney

Summary Histochemical and immunohistochemical staining techniques have been used to investigate the localization of hexokinase isoenzymes within rat kidney tissue. Hexokinase type I was shown to be the major isoenzyme present. It was located mainly in the thin and thick limbs of loops of Henle, in distal tubules and in the transitional or dark cells in the initial portions of collecting ducts. The smooth muscle cells of arteries and arterioles, peripheral nerves and the transitional epithelial cells lining the renal pyramid also contained large amounts of the isoenzyme while smaller quantities were present in glomeruli and in collecting tubules near the papillary tip. The distribution pattern obtained in tubular epithelia agrees well with that demonstrated in earlier microdissection studies. It is also consistent with the suggestion that glycolysis provides the majority of the energy fuelling the sodium transport mechanisms which form such an essential feature of the countercurrent urine concentration system present within the renal medulla.     

An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.     

Membrane charge-associated heterogeneity of B-lymphocytes from human peripheral blood as reflected by cell partition in two-polymer aqueous phases

Aqueous solutions of dextran and of poly-(ethylene glycol) when mixed give rise to immiscible aqueous-aqueous two-phase systems which, when buffered and rendered isotonic, are suitable for the separation by partition of cells based on subtle differences in selected membrane surface properties. Mononuclear cells from human peripheral blood were obtained on a Hypaque-Ficoll cushion and were then separated on the basis of size on a velocity sedimentation gradient at unit gravity. Lymphocytes obtained in this manner were subjected to countercurrent distribution (CCD) in a phase system which reflects membrane surface charge-associated properties. Cells in the different cavities of the extraction train were examined by fluorescent techniques utilizing goat antihuman IgM and anti-human IgD (either separately or mixed) and for their ability to form rosettes with a sheep erythrocyte-antibody-complement (EAC) complex. Results indicate that the highest percentage of fluorescing cells and EAC rosetting cells are under the left part of the distribution. B-lymphocytes are highly heterogeneous and consist of at least two distinct sub-populations not attributable to a difference in surface immunoglobulins. Experimental variation and error preclude, at present, a statement relating to the partial separability of IgM- and IgD-bearing cells. Conversely, the differences in surface charge-associated properties of IgM- and IgD-bearing cells, if they exist, must be small.     

Elution countercurrent distribution

The single withdrawal technique, here called elution countercurrent distribution, was applied for the first time on aqueous two-phase systems. The modifications necessary for elution countercurrent distribution of a thin-layer countercurrent distribution apparatus are described. The results from a test run with sulfuric acid showed good agreement between the experimental and the theoretical curves for elution countercurrent distribution. Thylakoid membrane vesicles from spinach chloroplasts were subjected to the elution countercurrent distribution. A gradient of NaCl in the eluting upper phase was used to elute the different fractions in order of their affinity for the moving upper phase. Inside-out thylakoid vesicles were resolved into two major and several minor fractions having different chlorophyll a/b ratios. Sonicated inside-out thylacoid vesicles were resolved into at least five different fractions with different chlorophyll a/b ratios. An increased resolution is obtained with elution countercurrent distribution compared to the fundamental process.     

Cell-cell affinity probed by countercurrent distribution in two-polymer aqueous phase systems

Aqueous solutions of dextran and of polyethylene glycol when mixed form immiscible liquid two-phase systems with a polyethylene glycol-rich top and a dextran-rich bottom. Such phases can be buffered and rendered isotonic and are suitable for the partition of cells. The partition coefficient of cells (i.e., their relative affinity for the top or bottom phase or their adsorption at the interface) depends on the polymer concentrations, on the ionic composition and concentration and, most sensitively, on their membrane surface properties. When two cell populations are mixed the partition coefficient of each is unaffected by the presence of the other population unless an interaction takes place between them. By countercurrent distribution of two cell populations, in a phase system selected such that the partition coefficient of one population is high (i.e., more cells in the top phase) and of the other low, one should be able to detect subtle interactions between cells in the two populations should they occur. Results of experiments with a model system consisting of human peripheral blood mononuclear cells and sheep red blood cells bear out the feasibility of this approach. At low ratios of sheep erythrocytes to mononuclear cells no interaction is apparent. With increasing ratios there is an increasing shift in the distribution curve of subpopulations of mononuclear cells (i.e., T-lymphocytes) as they interact with the sheep red cells. Substitution of rabbit red cells for sheep erythrocytes (at high ratios) reveals a small yet significant shift of a subpopulation of mononuclear cells as well. Distribution of human peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (all B-lymphocytes) are essentially unaffected by the presence of sheep red cells. This sensitive new method, still in its infancy, holds out the hope for the detection of previously unknown cell-cell affinities and for probing suspected cell-cell interactions.     

Membrane surface properties of reticulocytes from rats rendered severely anemic with phenylhydrazine as determined by partition in aqueous phase systems

Partition was studied in aqueous dextran-polyethylene glycol two-phase systems of reticulocytes from rats rendered severely anemic with phenylhydrazine.

1.

1. The countercurrent distribution of reticulocyte populations from phenylhydrazine-injected rats indicate that these are highly heterogeneous. The distribution patterns obtained include separation of such reticulocytes into two distinct sub-populations, a broad (and therefore obviously heterogeneous) curve, or a distribution intermediate between these.     

Uptake of 5-hydroxytryptamine by mast cells in vivo: a cytofluorometric study of mast cells and individual mast cell granules.

Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.