Separation of cells by velocity sedimentation

Abstract

A system for fractionating populations of Jiving cells by velocity sedimentation in the earth's gravitational field i s described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes pIace primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be signBcantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 -/hour) it is 1.5 X 1 0 ' cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Response of tumour-related and normal lymphocytes to antigens on fibroblasts from embryos of varying age

“…Cell preparation, irradiation, 51Cr-cytotoxicity assays and velocity sedimentation.-These have been described in detail in previous publications (Gorczynski, 1976a, c;Miller and Phillips, 1969).…”

Use of xantham gum to suspend large particles during flow cytometric analysis and sorting

“…In fact, the data shown in Figure 1 can be fit exactly to the Stokes equation by assuming a cell density of 1.067 gicm" (11) and an extracellular space fraction in the spheroid of 0.58 (3); this gives an effective spheroid density of 1.041 gicm". If corrections for density are made, the fit line to the data for spheroids in PBS shown in Figure 1 extrapolates to a value of 16.8 mm/h for a single cell of 15 pm diameter; this value is similar to the 15-18 mmih sedimentation velocities of single mammalian cells in suspension (12). The sedimentation velocity data indicate that a 100 pm diameter spheroid would require 66 min to settle the length of a standard flow sample tube (75 mm long) in a 0.1% xantham gum solution, versus 7.5 min in PBS alone.…”

Density/volume analysis in the study of cellular heterogeneity in human ovarian carcinoma

“…Sedimentation and volume analyses of cell populations have been applied in the past to normal and neoplastic haemopoietic tissues (Miller, 1973). The method described is conceptually different, in that it is based on the volume analysis of cell fractions prepared on a density gradient, and in practical terms the resolution with respect to density is greater.…”

“…Recovery was consistently >95%, and viability of the cells was not compromised. Velocity gradient8.-A modification of the procedure of Miller & Phillips (1969) was applied. About 107 cells from individual density fractions (pooled from multiple identical density gradients) were layered on top of linear gradients of 5-15% FCS (prepared in 50ml siliconized glass tubes).…”

“…Combined density/volume analysis-Cellvolume measurements on individual density or velocity sedimentation fractions were made with an electronic cell sizer, consisting of a 140jum Coulter volume orifice (Coulter Electronics, Hialeah, Florida) connected to custom signal-processing electronics (Miller, 1973). The cell-volume data were collected with a Nuclear Data (Schaumberg, Illinois) ND1200 Pulse Height analyser, and later transferred to a PDP 11/10 minicomputer (Digital Equipment Corporation, Maynard, Massachusetts) connected to a Tektronix Model 4662 Plotter (Beaverton, Oregon).…”