Centrifugal elutriation to obtain synchronous populations of cells.
Counterflow centrifugal elutriation is a noninvasive method for separating large numbers of cells on the basis of their size and mass. For mammalian cells, this method is useful for separating mixed populations of cells, in particular cells at different stages of the cell division cycle without perturbing cell metabolism or using synchronizing agents. This unit describes a method for separating 2 x 10(8) cells using the standard JE-6B rotor or larger numbers of cells in the JE-5.0 rotor. To verify the purity and to characterize the cell cycle positions of cells in the elutriated populations, the unit includes protocols for measuring nascent DNA synthesis by [(3)H]thymidine incorporation and for detecting DNA synthesis and content by propidium iodide flow cytometry alone or in combination with bromodeoxyuridine incorporation.
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Seattle Genetics, Inc., Bothell, Washington, USA.
Donaldson
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D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis; D045744:Cell Line, Tumor / Q000166:cytology; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D011419:Propidium / Q000032:analysis; D013194:Staining and Labeling; D013936:Thymidine / Q000378:metabolism; D014316:Tritium / Q000032:analysis
D005456:Fluorescent Dyes; D014316:Tritium; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine