From: Simon Hunt (simon.hunt@pathology.oxford.ac.uk)
Date: Wed Feb 05 2003 - 04:30:26 EST
RE: keeping cells in suspensionAs an alternative to the smart thixotropic properties of xantham gum, you can use a simple non-viscous isopycnic suspension medium, i.e. one whose density equals that of the cells. If you choose the right density, suspended cells neither sink nor rise. They just float where they are. When we sorted rat bone marrow cells we used 70% v/v Percoll (colloidal silica coated with polyvinyl pyrrolidone; Amersham) made up to isotonicity in a simple Dulbecco's A + B salt solution i.e. PBS containing divalent cations. This medium has the advantage of being no more viscous than any regular handling medium, so it's easy to resuspend your cells in it after the final pelleting following antibody staining. The reference is in the Methods section of: Dyer, M. J. and S. V. Hunt (1981). "Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity." J Exp Med 154(4): 1164-77. The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers ..... (for the curious, W3/13 would nowadays be called CD43) You have to compromise a tiny bit. Cell populations from ex vivo sources have slightly different densities, so a separation of the lighter ones from the denser ones becomes gradually noticeable after several hours. Conveniently, dead cells tend to float upwards so they are last through the SIP tube. It would be worth checking the best Percoll concentration for your particular cell type. It's a picnic, really! Simon Hunt Lecturer in Immunology, University of Oxford Dunn School of Pathology, South Parks Road, OXFORD OX1 3RE, U.K. http://users.path.ox.ac.uk/~svhunt
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