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Density separation of human red blood cells on self forming Percoll gradients: correlation with cell age
H U Lutz1, P Stammler, S Fasler
1Laboratory for Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, Zurich.
Human red blood cells were density separated on self-forming Percoll gradients. Redistribution of density fractionated red blood cells was studied by recentrifugation on self-forming Percoll gradients. A protocol that avoids centrifugation of red cells prior to removal of white cells and introduces EDTA before red cell pelleting completely avoided redistribution. Dense red cells separated according to this method were senescent on the basis of a biochemical and a physical criterion: the increase in the band 4.1a:4.1b ratio (Mueller, T., Jackson, C.W., Dockter, M.E. and Morrison, M. (1987) J. Clin. Invest. 79, 492-499) and the loss of maximum deformability. Characterization also included the relative content of two surface proteins (complement receptor 1, CR1 (Ripoche, J. and Sim, R.B. (1986) Biochem. J. 235, 815-821); decay accelerating factor, DAF) on density fractionated red cells. Unlike cytoplasmic proteins, these proteins face similar conditions, whether located on circulating reticulocytes or aging red cells. Both components were lost linearly within experimental errors with cell density and were lower by 60 and 40% in dense than light cells, respectively.
Human red blood cells were density separated on self-forming Percoll gradients. Redistribution of density fractionated red blood cells was studied by recentrifugation on self-forming Percoll gradients. A protocol that avoids centrifugation of red cells prior to removal of white cells and introduces EDTA before red cell pelleting completely avoided redistribution. Dense red cells separated according to this method were senescent on the basis of a biochemical and a physical criterion: the increase in the band 4.1a:4.1b ratio (Mueller, T., Jackson, C.W., Dockter, M.E. and Morrison, M. (1987) J. Clin. Invest. 79, 492-499) and the loss of maximum deformability. Characterization also included the relative content of two surface proteins (complement receptor 1, CR1 (Ripoche, J. and Sim, R.B. (1986) Biochem. J. 235, 815-821); decay accelerating factor, DAF) on density fractionated red cells. Unlike cytoplasmic proteins, these proteins face similar conditions, whether located on circulating reticulocytes or aging red cells. Both components were lost linearly within experimental errors with cell density and were lower by 60 and 40% in dense than light cells, respectively.