Buoyant Density Separation with Linear Gradients of Bovine Serum Albumin and Analysis by Centrifugal Cytology and Flow Techniques

Abstract

The last review of the work of my laboratory (Leif, 1970a) described the technique for buoyant density separation of cells in isotonic gradients of bovine serum albumin (BSA) and the application of that technique to the separation of human and rabbit erythrocytes. The various gradient media, BSA, Ficoll, iodothalamate, and colloidal silica, were reviewed. The method of preparation of the BSA, including deionization, filtration, and addition of salts and tissue culture media, were described in detail. The biological studies presented in that review were limited to the first class of cells studied, erythrocytes (human and rabbit). Briefly, reproducible buoyant density distributions of human erythrocytes were obtained, and individual fractions were shown to be capable of being rebanded. The average buoyant density of the erythrocytes from four individuals was 1.0808 ± 0.0004 g cm-3. The average density from a fifth individual was 0.0028 g cm-3 greater. The entire buoyant density distribution shifts with salt content; the erythrocytes were shown to behave as “perfect” osmometers. Salt gradients were shown to spread or narrow the distribution predictably, and it was suggested that these gradients might be used to increase the resolving power of the density separations.