Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.
This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.
Centre for Cancer Research, Westmead Millennium Institute for Medical Research and University of Sydney.
Robert
R
Welschinger
Centre for Cancer Research, Westmead Millennium Institute for Medical Research and University of Sydney; linda.bendall@sydney.edu.au.
Linda J
LJ
Bendall
D001973:Bromodeoxyuridine / Q000493:pharmacokinetics; D002453:Cell Cycle / Q000502:physiology*; D059447:Cell Cycle Checkpoints; D002455:Cell Division / Q000502:physiology; D045744:Cell Line, Tumor; D058948:Cell Tracking / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism / Q000473:pathology; D016196:S Phase / Q000187:drug effects
D004247:DNA; D001973:Bromodeoxyuridine