ProtocolReanalysis of the protocol for in vitro synchronization of mammalian astrocytic cultures by serum deprivation
Section snippets
Commentary
It has been proposed that it is possible to synchronize astrocytes obtained from newborn rat brains by whole-culture treatment of cells [2]. The proposed method was to first starve cells in low serum, then refeed the cells with serum to obtain a synchronized culture. There are two parts to the detailed protocol: (1) proliferation of astrocytes under optimal conditions in vitro until the cells reach the desired confluence; and (2) synchronization of cultures by serum removal and proposed arrest
Discussion
The interest in the study of events during the cell cycle has led to numerous proposals for methods to synchronize cells. Here, one published method is analyzed. It is concluded that the published method neither presented evidence that the cells are synchronized nor considered theoretical arguments that the whole-culture method used–serum starvation followed by refeeding serum–does not, and cannot, synchronize cells [12].
Lest it be thought that it is proposed here that cell synchronization is
Acknowledgments
This work was supported by Grant MCB-0323346 from the National Science Foundation. Additional support for this research came (in part) from the National Institutes of Health through the University of Michigan's Cancer Center Support Grant (5 P30 CA46592).
References (21)
- et al.
A genome-wide transcriptional analysis of the mitotic cell cycle
Mol. Cell
(1998) - et al.
In vitro synchronization of mammalian astrocytic cultures by serum deprivation
Brain Res. Protoc.
(2003) Reappraisal of G1-phase arrest and synchronization by lovastatin
Cell Biol. Int.
(2002)Whole-culture synchronization can not, and does not, synchronize cells
Trends Biotechnol.
(2004)On G0 and cell cycle controls
Bioessays
(1987)G1 and S phase gene expression cannot be analyzed in mammalian cells synchronized by inhibition
Microb. Comp. Genomics
(1997)Mammalian cells are not synchronized in G1-phase by starvation or inhibition: considerations of the fundamental concept of G1-phase synchronization
Cell Prolif.
(1998)On the proposal of a G0 phase and the restriction point
FASEB J.
(1998)The continuum model and G1-control of the mammalian cell cycle
Prog. Cell Cycle Res.
(2000)Minimally disturbed, multi-cycle, and reproducible synchrony using a eukaryotic “baby machine”
Bioessays
(2002)
Cited by (11)
Invariant mRNA and mitotic protein breakdown solves the Russian Doll problem of the cell cycle
2009, Cell Biology InternationalCitation Excerpt :First, the clear presence of cell-cycle variation in protein content does not mean that cyclical mRNA variation is expected. Second, the data on mRNA variation during the cell cycle has to be reconsidered, with attention to problems of synchronization of cells and perturbations when whole-culture methods are used (Cooper, 1998c, 2002, 2003a,b, 2004c,d, 2005, 2006), as well as problems with microarrays (Cooper and Shedden, 2003; Shedden and Cooper, 2002a,b). And finally, one must consider the logical and theoretical problems with postulating mRNA variation during the cell cycle as exemplified by both the infinite regression problem and the minor affect of mRNA variation on protein variation.
Chapter 12 Phosphoproteomics
2008, Comprehensive Analytical ChemistryCitation Excerpt :Starvation is also thought to partly synchronize the cells in G0/G1 phase of the cell cycle. Cells subjected to serum starvation will have a wide distribution of cell sizes, proteomes and content of DNA, and will therefore reflect cells of a specified stage in the division cycle [53,54]. It is thought that after starvation each cell will respond equally to the subsequent external stimulation.
A kinetic model for calcium dynamics in RAW 264.7 cells: 2. Knockdown response and long-term response
2007, Biophysical JournalCitation Excerpt :This can be achieved by arresting the cells in a specific phase using appropriate pharmacological inhibitors or through serum deprivation, which would arguably not interact with the KD of interest; see Davis et al. (20) and Chou and Langan (21) for a review. Cooper (22,23) has discussed potential drawbacks of a serum deprivation method. With cell cycle synchronization in place, the response for a chosen dose of C5a in two different control cell lines should be nearly the same, provided the number of cells is large in both batches; any difference can only be attributed to experimental errors.
Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe
2020, Neural Regeneration ResearchPhenotypic Assays to Identify Agents That Induce Reactive Gliosis: A Counter-Screen to Prioritize Compounds for Preclinical Animal Studies
2015, Assay and Drug Development Technologies