Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes. Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic 59Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

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.     

Alterations in membrane surface properties during cell differentiation as measured by partition in aqueous two-polymer phase systems: Rat intestinal epithelial cells

Membrane surface properties of rat intestinal epithelial cells (crypt base to villus tips) were studied by cell partition in a two-polymer aqueous phase system. A higher partition generally reflects higher cell surface charge (or charge-associated properties) which is not necessarily the same as the charge determined by cell electrophoresis since the latter reflects only the charge at the plane of shear while the former gauges it deeper into the membrane [10]. Cells were prepared by the method of Weiser [22] which sequentially yields cell fractions from villus tips to crypt base. The isolated cells were subjected to countercurrent distribution in a dextran-polyethylene glycol aqueous two-phase system. Countercurrent distribution on the first fractions obtained by Weiser's method have a peak to the left and a smaller peak to the right indicating a surface membrane heterogeneity of upper villus cells; last fractions have a peak only to the right. When all fractions are pooled before countercurrent distribution two well-separated peaks are obtained with the right peak sometimes showing additional heterogeneities. Experiments combining isotope labeling of cells with countercurrent distribution lead us to conclude that the membrane charge (or charge-associated properties) of crypt base cells increases during differentiation and that the charge of the villus cells to which they give rise then diminishes during maturation. The charge of the bulk of the upper villus cells is the lowest of any in the intestinal cell population. The basis for the alteration in charge has not been established but the phenomenon of changing membrane surface charge (or charge-associated properties) as a function of cell differentiation, maturation and aging appears to be a general phenomenon having been found and traced in different cell populations [14, 16, 17, 28].     

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.     

Differential effect of neuraminidase-treatment on the surface charge-associated properties of rat reticulocytes and erythrocytes: Studies by partitioning in two-polymer aqueous phases

Rat reticulocytes undergo charge-associated surface changes, detectable by cell partitioning in charged dextran-poly(ethylene glycol) aqueous phase systems, as they become mature erythrocytes. Young reticulocytes have a lower partition coefficient, i.e., quantity of cells in the top phase as a percentage of total cells added, than do mature erythrocytes. Sialic acid is the main charge-bearing group on red blood cells and, in the case of the rat, most of the sialic acid can be removed by treatment of the cells with neuraminidase (Vibrio cholerae). By combining isotopic ⁵⁹Fe-labeling of reticulocytes with countercurrent distribution of the entire red blood cell population in charged dextran-poly(ethylene glycol) aqueous phases we have now studied the relative effect of neuraminidase-treatment on rat reticulocytes and mature erythrocytes. It was found that neuraminidase-treatment (a) does not eliminate surface differences, detectable by partitioning, between rat reticulocytes and erythrocytes and (b) reduces the partition coefficient of mature erythrocytes to a greater extent than the partition coefficient of reticulocytes indicating a differential effect of this enzyme on the two cell populations.     

Effect of membrane cholesterol enrichment or depletion on the partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases

It has previously been shown that by appropriate manipulation of polymer concentrations and ionic composition and concentration one can select whether charge-associated or lipid-related membrane surface properties are reflected by cell partition in dextran-poly(ethylene glycol) aqueous two-phase systems (Walter, H. (1977) in Methods of Cell Separation ((Catsimpoolas, N., ed.), Vol. 1, pp. 307–354, Plenum Press, New York). In the current experiments we have studied the partition behavior of human erythrocytes and found that not only lipid-related but also charge-associated membrane properties are altered as a consequence of cholesterol-enrichment or -depletion. Results further indicate that, just as cell partition in charged phase systems reflects membrane charge-associated properties not readily measured by means other than partition (Brooks, D.E., Seaman, G.V.F. and Walter, H. (1971) Nat. New Biol. 234, 61–62; Walter, H., Tung, R., Jackson, L.J. and Seaman, G.V.F. (1972) Biochem. Biophys. Res. Commun. 48, 565–571), cell partition in uncharged phases reflects membrane lipid-related properties also not readily measured by other means.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems.

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging.The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

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.     

The effect of neuraminidase on the relative surface charge-associated properties of rat red blood cells of different ages

Approx. 70% of the sialic acid on the rat erythrocyte surface is susceptible to cleavage by neuraminidase (Vibrio cholerae). Neuraminidase treatment results in a reduction in the partition coefficient (K) of the red cells in a charged dextran-poly(ethylene glycol) aqueous phase system and in the electrophoretic mobility of the cells. Countercurrent distribution of rat neuraminidase-treated erythrocytes, containing 59Fe-labeled mature red cells of distinct age, indicates that (a) the electrophoretic mobilities of red cells in different cavities along the extraction train increase with increasing K, as is the case with untreated erythrocytes, and (b) the cell age-related differences in surface charge-associated properties are neither eliminated nor altered by the enzyme action.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with⁵¹Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations⁵¹Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning⁵¹Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phopshate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By “balancing” the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseqarability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.     

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.     

Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil

Phagocytosis of Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in human mononuclear phagocytes and causes Legionnaires' disease, occurs by a novel mechanism. A phagocyte pseudopod coils around the bacterium as the organism is internalized. Human monocytes, alveolar macrophages, and polymorphonuclear leukocytes all phagocytize L. pneumophila by this unusual process, termed "coiling phagocytosis," and these leukocytes phagocytize not only live L. pneumophila in this way, but also formalin-killed, glutaraldehyde-killed, and heat-killed L. pneumophila. In contrast, under the same experimental conditions, monocytes phagocytize Streptococcus pneumoniae, encapsulated and unencapsulated E. coli, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Neisseria gonorrhoeae, and Neisseria meningitidis by conventional phagocytosis. Treatment of L. pneumophila with high-titer anti-L. pneumophila antibody abolishes coiling phagocytosis; such bacteria are internalized by conventional phagocytosis. These experiments raise the possibility that a surface component of L. pneumophila mediates the unusual response by the phagocyte. Such a component, if elaborated in vivo, might be responsible for extrapulmonary manifestations of Legionnaires' disease suspected of being toxin-mediated.     

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.     

Membrane surface properties of sheep erythrocytes, an immunological reagent, after different treatments as reflected by partition in two-polymer aqueous phases.

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as "immunological reagents" to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueous-phase systems selected so as to reflect charge-associated or lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient, K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increased Ks and unchanged Ks, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reduced Ks of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.     

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.     

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.