THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.