Homogeneity of antibody-producing cells as analysed by their buoyant density in gradients of Ficoll

Abstract

Ficoll, a polymer of sucrose, has several advantages over proteins for preparation of density gradients used to study mammalian cells. In addition to its ease of preparation, Ficoll as a result of being an uncharged molecule, does not bind ions from solution and, therefore, does not affect the osmolality of the medium in which it is dissolved.

In studies on antibody-producing cells we found that the measurement of buoyant density in Ficoll at pH 7.2 gives an accurate measurement of cell density. In such gradients antibody-producing cells band in a single peak at a density of 1.070 g/cm³. Two factors have a profound influence on the density profile of antibody-producing cells. Lowering the pH to 5.5 lowers their buoyant density to 1.062 g/cm³. Creation of an inverse osmotic gradient varying from 294 m-osmolal at the top of the gradient to 286 m-osmolal at the bottom splits the single peak of antibody-producing cells into three distinct peaks. This effect of osmotic gradients could account for the multiple peaks observed when cells are separated on protein gradients.