应用自由流电泳分离小鼠脾淋巴细胞

自由流电泳具有独特的全液相自由分离过程和非常温和的分离条件,使用这种技术进行细胞等水下溶性活性物质的分离不仅效率高,而且可以较好地保存其生物活性和成活力,自由电泳在低离子强度的三乙醇胺缓冲液系统中进行了Ｂ和Ｔ淋巴细胞的分离,并利用反向介质技术使分离后的细胞处于生理盐溶液中。     

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.     

The microfibrillar proteins of human hair: separation by high-performance liquid chromatography and isolation of some proteins enriched in glycine and tyrosine

The microfibrillar proteins of human hair have been studied by reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A chromatographic procedure which isolates the microfibrillar proteins from other hair-matrix proteins and separates them into collectable fractions has been introduced. These fibrous proteins fall into two major subgroups which are resolved into six components. The same procedure has also resulted in the identification and simultaneous separation of a group of proteins rich in glycine and tyrosine never before detected in human hair. Comparative electrophoretic studies of the crude microfibrillar proteins reveal five bands with apparent molecular weights of 47,000, 50,000, 53,000, 57,000, and 62,000. The relationship between the electrophoretic bands and the chromatographic fractions is now under investigation.     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

Preparative Electrophoretic Separation of Antibody Forming Cells

A procedure is described for preparative electrophoretic separation of lymphoid cells. The separations were performed with a free flow electrophoretic cell separator model VAP IV (Desaga, Heidelberg, Bender & Hobein, Munich, Brinkmann Instruments, Westbury, N. Y.). Rats were immunized with sheep erythrocytes (SRBC), lymph node cells electrophoretically separated at different times after immunization and the fractions obtained subsequently cultured in diffusion chambers. The antibody forming cells and the morphological composition of the fractions was determined after separation and after culture. Lymph node cells could be separated into 16 fractions. Within this heterogeneous distribution profile two narrow distributions of antibody forming cells of the same specificity but of different stages of development could be detected. The distribution of lower electrophoretic mobility contained the primed lymphocytes and blast cells, the faster distribution contained the differentiated plasma cells. It was found that a homogeneous cell population is rather homogeneous in its electrophoretic mobility. This is an indication that the electrophoretic mobility would be a useful parameter enabling separation of functionally defined cell populations.     

Preparative free flow electrophoresis for the isolation of two populations of proximal cells from the rabbit kidney

High voltage free flow electrophoresis is a carrier-free method used for analytical and preparative cell separation, based on charge surface properties of cells. Two cell populations from the proximal tubule of the rabbit kidney were isolated by free flow electrophoresis from a suspension of pure proximal cells. This single-cell suspension was obtained through an original method by the combination of a Ca-binder action and gentle mechanical treatment associated with several shifting steps, on a pure suspension of isolated proximal tubules. Before the electrophoretic separation, the proximal cell origin was confirmed by enzymatic marker measurements, and the metabolic capacity was assessed by the cell respiratory activity. The isolated cells were very poor in distal tubule marker enzymes and were enriched in proximal tubule marker enzymes. Respiratory measurement showed a high cell metabolic capacity. After the electrophoretic separation, the origin of the cell populations was assessed by measuring specific marker enzymes. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and a low glucose-6-phosphatase activity. The fast moving cells showed a high glucose-6-phosphatase content and a poor gamma-glutamyl transpeptidase activity. Cells isolated by free flow electrophoresis were shown to possess long microvilli. This new methodology, allowed for the first time, the separation of a fast-moving cell population originating from the convoluted portion of the proximal tubule and a slow-moving cell population originating from the straight part of the proximal tubule of the rabbit kidney.     

STUDIES ON SALT ACTION : X. THE INFLUENCE OF ELECTROLYTES UPON THE VIABILITY AND ELECTROPHORETIC MIGRATION OF BACTERIUM COLI.

1. The strain of Bacterium coli used in these experiments multiplies in distilled water at pH 6.0 and pH 8.0 and in Ringer-Locke solution at pH 6.0. Under all the other conditions studied the numbers decrease with the passage of time. 2. The electrophoretic charge of the cells is highest in distilled water at pH 6.0 and pH 8.0. Under all other conditions studied the velocity of migration is decreased, but the decrease is immediate and is not affected by more prolonged exposure. 3. A strongly acid solution (pH 2.0) causes a rapid death of the cells and a sharp decrease in electrophoretic charge, sometimes leading to complete reversal. 4. A strongly alkaline solution (pH 11.0) is almost as toxic as a strongly acid one, although in distilled water the organisms survive fairly well at this reaction. Electrophoretic charge, on the other hand, is only slightly reduced in such an alkaline medium. 5. In distilled water, reactions near the neutral point are about equally favorable to both viability and electrophoretic charge, pH 8.0 showing slightly greater multiplication and a slightly higher charge than pH 11.0. In the presence of salts, however, pH 8.0 is much less favorable to viability and somewhat more favorable to electrophoretic charge than is pH 6.0. 6. Sodium chloride solutions, in the concentrations studied, all proved somewhat toxic and all tended to depress electrophoretic charge. Very marked toxicity was, however, exhibited only in a concentration of .725 M strength or over and at pH 8.0, while electrophoretic migration velocity was only slightly decreased at a concentration of .0145 M strength. 7. Calcium chloride was more toxic than NaCl, showing very marked effects in .145 M strength at pH 8.0 and in 1.45 M strength at pH 6.0. It greatly depressed electrophoretic charge even in .0145 M concentration. 8. Ringer-Locke solution proved markedly stimulating to the growth of the bacteria at pH 6.0 while at pH 8.0 it was somewhat toxic, though less so than the solutions of pure salts. It depressed migration velocity at all pH values, being more effective than NaCl in this respect, but less effective than CaCl₂. 9. It would appear from these experiments that a balanced salt solution (Ringer-Locke''s) may be distinctly favorable to bacterial viability in water at an optimum reaction while distinctly unfavorable in a slightly more alkaline solution. 10. Finally, while there is a certain parallelism between the influence of electrolytes upon viability and upon electrophoretic charge, the parallelism is not a close one and the two effects seem on the whole to follow entirely different laws.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

Use of Hydrocyclones for Mammalian Cell Retention: Separation Efficiency and Cell Viability (Part 1)

A hydrocyclone with a volume of 2.56 cm³ was studied as a potential cell retention device for mammalian cell cultures (6 L volume). For the feasible operation range (0.9 to 1.6 L/min flow corresponding to pressure drops of 0.4 to 1.3 bar) the hydrocyclone was characterized with regard to flow split (underflow‐to‐overflow ratio) and flow ratio (underflow to supply). Cultures of BHK and HeLa cells (with low cell concentrations) were applied to measure separation efficiency and cell viability for a hydrocyclone operation period of 3 min corresponding to a cell suspension throughput of 2.7 to 4.8 L. Cell separation efficiencies ranged from 0.77 to 0.97 and cell viability was not affected except for BHK cells in the overflow at the highest pressure drop (1.3 bar). As the overflow is commonly used for product harvest and cells are discarded, the application of the hydrocyclone has no detrimental effect on the reactor perfusion system. The results indicate that only cells passing from the primary vortex downwards into the inner secondary vortex and from there upwards could be damaged. Evidence for this hypothesis is obtained from operating the hydrocyclone with closed overflow (only centrifugal forces acting) for a period of 3 h. In these studies no significant effect on cell viability could be detected for HeLa and CHO cells. Hence, the results indicate that the hydrocyclone can be appropriately used for cell retention and separation in perfusion cultures. Application at higher pressures is recommended whereby separation efficiencies of 0.97 without any loss in viability can be achieved.     

The lateral separation of contacts on erythrocytes agglutinated by polylysine.

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10-225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 microns as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially periodic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons

In the present study, human NT2 neurons obtained from embryonic teratocarcinoma (NT2) cells were established as human in-vitro model to investigate the mechanisms associated with hypoxia/ischemia-induced neuronal injury. NT2 neurons express functional NMDA receptors that are of particular significance for hypoxia/ischemia-related neuronal damage. In patch-clamp recordings under normoxic conditions, NMDA (plus 10 microM glycine)-induced inward currents (EC(50)=43.7 microM) were distinctly antagonized by memantine, a blocker of the receptor channel, but only slightly by 5,7-dichlorokynurenic acid (DCKA), a glycine(B) binding site antagonist. Immunohistochemistry demonstrated that the NT2 neurons are mostly GABAergic; they predominantly express the NMDA receptor subunits NR2B and NR2C, and lower levels of NR1 and, particularly, of NR2A. Upon glucose and oxygen deprivation for 3h the loss of cell viability measured directly after 3h was higher than after application of either hypoxia or aglycemia as assessed by propidium iodide flow cytometry. Ischemic conditions significantly reduced the NMDA responses associated with a decrease in EC(50) and decreased mitochondrial membrane potential as detected by JC-1 flow cytometry. Memantine (50 microM) and CGS19755 (a competitive NMDA receptor antagonist; 10 microM) reduced ischemia-induced cell death, in contrast to DCKA (10 microM). In conclusion, in the present human in-vitro model for studying the molecular mechanisms associated with ischemic injury, neuroprotection could be achieved with NMDA receptor antagonists but not with a glycine(B) binding site antagonist. Accordingly, glycine antagonists might not represent an optimal therapeutic strategy for preventing ischemic neuronal damage in contrast to NMDA receptor antagonists like memantine.     

Free-flow electrophoresis. I. Theoretical and experimental investigations of the influence of mechanical and electrokinetic variables on the efficiency of the method.]

In the present paper, various types of band-broadening effects in free-flow electrophoresis were investigated. They resulted from the velocity profiles of the liquid curtain and electroosmosis, temperature gradient, thermal diffusion and sample inlet geometry. An analytical free-flow electrophoresis apparatus permitted easy observation of these parameters. The experiments showed that the influence of the temperature gradient was negligible, whereas the effect of the velocity profiles on band broadening was higher than theoretically expected. In preparative work this is of utmost importance, since overlap of bands is not desirable. In this case, the zeta potential of the walls can be adjusted to that of the material to be separated, resulting in a considerable reduction of band broadening. Various approaches are indicated. Further attention was given to the influence of the relaxation effect on the separation. Conditions are shown where particles are separated either according to their surface charge density or to their size. The practical relevance of the results is discussed.     

Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism

Background & Aims

Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.

Methods

Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.

Results

Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.

Conclusion

In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.     

Use of glycine as a non-enzymic procedure for separation of mouse embryonic tissues and dissociation of cells

A non-enzymic procedure for separation and cell dissociation of mouse embryonic tissues is described, utilizing solutions of either 1 or 0.5 M glycine with EDTA. Incubation of blastocysts in glycine resulted in decompaction of cells without complete dissociation. Post-implantation egg cylinders were readily separated into component tissue layers, with subsequent dissociation into single cells. Cell viability was high after such treatment, and all cell types adhered rapidly and spread out on plastic culture dishes. Parietal endoderm, visceral yolk sac and amnion tissues from 10th and 14th day embryos were incubated in glycine, with resulting cell dissociation and/or tissue separation. Changes in cell adhesion properties appear to take place during development of these tissues. PSA1 embryoid bodies readily separated into the outer ‘endoderm’ layer and inner embryonal carcinoma cell core after a brief incubation in glycine. However, this procedure was unsuccessful for dissociation of cells from a plastic or gelatin substrate in vitro, suggesting that cell-substrate adhesion in vitro may differ from that in vivo.     

Cell inactivation and membrane damage after long-term treatments at sub-zero temperature in the supercooled and frozen states

The survival of cells subjected to cooling at sub-zero temperature is of paramount concern in cryobiology. The susceptibility of cells to cryopreservation processes, especially freeze-thawing, stimulated considerable interest in better understanding the mechanisms leading to cell injury and inactivation. In this study, we assessed the viability of cells subjected to cold stress, through long-term supercooling experiments, versus freeze-thawing stress. The viability of Escherichia coli, Saccharomyces cerevisiae, and leukemia cells were assessed over time. Supercooled conditions were maintained for 71 days at -10 degrees C, and for 4 h at -15 degrees C, and -20 degrees C, without additives or emulsification. Results showed that cells could be inactivated by the only action of sub-zero temperature, that is, without any water crystallization. The loss of cell viability upon exposure to sub-zero temperatures is suggested to be caused by exposure to cold shock which induced membrane damage. During holding time in the supercooled state, elevated membrane permeability results in uncontrolled mass transfer to and from the cell maintained at cold conditions and thus leads to a loss of viability. With water crystallization, cells shrink suddenly and thus are exposed to cold osmotic shock, which is suggested to induce abrupt loss of cell viability. During holding time in the frozen state, cells remain suspended in the residual unfrozen fraction of the liquid and are exposed to cold stress that would cause membrane damage and loss of viability over time. However, the severity of such a stress seems to be moderated by the cell type and the increased solute concentration in the unfrozen fraction of the cell suspension.     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

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 selective 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 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) 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.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.