Ann L. Griffith

Electrophoretic separation of cells in a density gradient

Abstract A bulk electrophoretic method is described for the separation of mammalian cells in Ficoll-sucrose density gradients. The fractionation of cells is performed in a simple commercially available apparatus (Buchler Poly-Prep) which should facilitate the application of the technique in various laboratories. Details of the experimental procedure are presented along with typical model separations of erythrocytes from various species and mouse lymphocytes.

Electrophoresis with continuous scanning densitometry: separation of cells in a density gradient.

Abstract An instrument and procedure for electrophoresis with continuous optical scanping densitometry, automated data processing, and related methodology are described for the continuous analysis of electrophoresis and unity gravity sedimentation of macromolecules or cells in a static density gradient system. The instrument consists of a dual-beam spectrophotometer, a scanning stage and scanner control unit, an electrophoresis cell cassette, a filling/purging/cooling module, an analog-to-digital converter, and a digital data-logger. The distribution of cells is monitored repetitively during migration by absorbance measurements at any wavelength in the 200–800-nm range. A computer program provides the statistical analysis of each peak (baseline correction, smoothing, area, mean, standard deviation, skewness, and kurtosis) which can be further utilized for computing additional parameters, such as resolution and heterogeneity. A mixture of human and rabbit erythrocytes were used as a model system to evaluate the performance of the instrument and demonstrate some of its capabilities.

Density gradient electrophoresis of mouse spleen lymphocytes. Separation of t and b cell fractions.

Preparative electrophoresis in an isotonic Ficoll--sucrose density gradient has been employed for the separation of mouse (C57Bl/6J) spleen lymphocyte subpopulations. The separated cells were pooled into six fractions according to their relative position (Rp) within the total cell distribution. In general, the high mobility cells were identified as T lymphocytes. These cells exhibited immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated mouse anti-theta globulin and responded in vitro to phytohemagglutinin stimulation. The low mobility cells were activated in vitro by E. coli lipopolysaccharide and showed immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated anti-mouse Ig which is typical of mouse B lymphocytes. Both T and B cells were completely isolated from each other in certain fractions of very high and very low mobility, respectively. Overlapping of the two distributions was observed in the intermediate mobility fractions. The method which utilizes an inexpensive commercially available apparatus should be useful for the preparation of other lymphocyte subpopulations differing in surface charge.

Transient state isoelectric focusing: effects of zone load and carrier ampholyte concentration on the kinetics of defocusing and refocusing in sucrose density gradients.

Abstract The kinetics of focusing, defocusing, and refocusing of l -histidyl- l -tyrosine in a sucrose density gradient have been studied utilizing a special apparatus for repetitive scanning of the isoelectric focusing column, employing uv absorption optics and a digital data acquisition system. Starting from a uniform or triangular concentration profile, the band is “focused” and a nearly linear pH gradient is formed during initial focusing. The electrical field is then abolished and free diffusion occurs. The electrical field is then reapplied (“refocusing”) and the band is allowed to sharpen, presumably reapproaching a steady state. The band width was measured quantitatively as the second moment about the mean (square of the standard deviation, σ2). In theory, measurements of σ2 versus time permit the estimation of the apparent diffusion coefficient (D) and the isoelectric focusing parameter (pE). If the electrical field strength E and the pH gradient, d( pH ) dx , were also measured, then one could calculate the slope of the pH mobility curve of the protein dM d( pH ) evaluated at the isoelectric point. D can be measured during the defocusing stage, and pE, D pE , or D can be measured during focusing or refocusing. Several limitations and difficulties in the verification of this theory have been encountered: First, the apparent diffusion coefficient depends on zone load in approximately a linear fashion. Accordingly, it is necessary to measure D at several zone loads, and then extrapolate to zero load by linear regression techniques. Second, the ampholyte concentration has a marked effect on both D and pE. Here we have no a priori reason to extrapolate to zero ampholyte concentration. Also, at present we have no satisfactory method for measurement of E. These preliminary studies should be helpful in indicating further directions for experimental refinement and for generalization of theory.

Differential 51Cr uptake of human peripheral lymphocytes separated by density gradient electrophoresis

Abstract Human peripheral lymphocytes were labeled with 51Cr before or after separation by preparative density gradient electrophoresis. In both cases, wide variations in the distribution of 51Cr in the electrophoresed cells was observed. In general, there was significantly more 51Cr per cell in the high mobility fractions. These results suggest caution in the interpretation of cytotoxic assays where 51Cr-labeled lymphocytes are used as target cells and prompt further studies by different separation methods.

Preparative Density Gradient Electrophoresis and Velocity Sedimentation at Unit Gravity of Mammalian Cells

In 1975 we described a new method for the preparative separation of mammalian cells by density gradient electrophoresis (Catsimpoolas and Griffith, 1975; Griffith et al., 1975). This development came about by combining the use of an isoosmolar Ficoll-sucrose density gradient medium (Boltz et al., 1973, 1976) with a commercially available apparatus originally designed for polyacrylamide gels (Jovin et al., 1964) and a fraction collection method involving differential pumping velocity of the chase and density gradient fluids (Svendsen, 1972). The technique can be used to separate highly viable and functional mammalian cells—in bulk quantities, i.e., up to 108 cells—if they exhibit different surface charge and therefore electrophoretic mobility. This capability has been demonstrated in several recent reports from this laboratory (Catsimpoolas et al., 1976a; Ault et al., 1976; Platsoucas et al., 1976; Griffith et al., 1976).

Transient velocity sedimentation of cells at unit gravity

Abstract A new kinetic method (TRANS-VELS) is described which allows for the first time the accurate determination of the sedimentation velocity of cells at unit gravity. This is accomplished by repetitive optical scanning of the cell distribution as a function of time and during transport through a shallow density gradient. Computer analysis of the statistical moments of the distribution is utilized for the measurement of the sedimentation velocity, its dispersion and the expected resolution. The latter two parameters being strongly time dependent have been estimated for the first time from kinetic data and bear important implications in the widely practiced preparative separation of cells by velocity sedimentation at unit gravity.

Transient electrophoretic and velocity sedimentation analysis of lymphocytes

A new instrument system (trans-analyzer) and procedures are described for the separation and characterization of lymphocytes by transient-state electrophoresis (trans-el), isoelectric focusing (trans-if), and velocity sedimentation at 1g (trans-vels) in iso-osmolar density gradients. Monitoring of cell migration in the electrical and 1g gravitational fields is performed in situ and repetitively by “absorbance” (light extinction) and scattering measurements. Data acquisition, processing, and display are performed by a computer. Kinetic analysis of the cell distribution provides precise information on the electrophoretic mobility and sedimentation velocity of lymphocytes under standardized conditions. Selective applications of these methods to model systems involving mouse (C57BL6J) thymus and spleen lymphocytes and human peripheral lymphocytes are reported. The availability of these techniques provides a new approach to immunophysical studies concerning the surface charge and hydrodynamic properties of immunocompetent cells.

Transient velocity sedimentation at unit gravity of human erythrocytes.

Abstract A new biophysical method, transient velocity sedimentation (TRANSVELS) at unit gravity, has been applied to the study of human erythrocytes from normal subjects and from patients with diseased states producing abnormal red blood cells. The hydrodynamic behavior of the cells during sedimentation was examined by repetitively recording the distribution profile as a function of time by the use of optical scanning methods. Computer analysis of the distributions allowed precise measurement of the mean sedimentation velocity ( s ) and the mean skewness ( ± S ) of peak profile. A two-dimensional plot of s versus S indicated significant differences between “normal” and “abnormal” erythrocytes by linear categorization techniques and probability density ellipses at the 0.1 density level. It is concluded that precise measurement of these two new parameters offers a new and valuable approach to the physical characterization of erythrocytes.

Transient Electrophoretic and Sedimentation Analysis of Cells in Density Gradients

Separation, identification, and characterization of mammalian cells represents one of the great challenges of present-day biological research. The elucidation of some of the most intricate problems in immunology, cell and molecular biology, and cancer research depends on new developments in the cell separations field. However, the diversity of mammalian cell properties can act both as a deterrent and as an enhancer of progress in this area. Therefore the coordinated utilization of physical, morphological, biological, and immunological characteristics of cells and their surfaces is necessary to accomplish the task of separation and analysis.