synchronize cell cycle in culture

Based on the knowledgeable wikipedia: “Several methods can be used to synchronise cell cultures by halting the cell cycle at a particular phase. For example, Serum starvation [10] and treatment with Thymidine or Aphidicolin [11] halt the cell in the G1 phase, Mitotic shake-off, treatment with colchicine [12] and treatment with Nocodazole [13] halt the cell in M phase and treatment with 5-fluorodeoxyuridine halts the cell in S phase.”

More specifically, for the U2OS cells, “HeLa and U2OS cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C in 5% CO2. To obtain mitotically synchronized HeLa cells, cells were treated with 100 ng/ml nocodazole for 16 h. To synchronize U2OS cells, cells were either blocked with 0.3 mM mimosine for 16 h to arrest at G1 phase or 100 ng/ml nocodazole for 16 h to arrest at M phase. Incubation with 4 mM hydroxyurea for 40 h results in U2OS cells arrested at S phase.”
It is based on the following paper:

Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8672-6.

Activation of Cdc2/cyclin B and inhibition of centrosome amplification in cells depleted of Plk1 by siRNA.
Liu X, Erikson RL.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

The events of the cell cycle, the stages at which the cell proliferates and divides, are facilitated and controlled by multiple signaling pathways. Among the many regulatory enzymes that contribute to these processes is the polo-like kinase (Plk). Plks have been reported to mediate multiple mitotic processes, including bipolar spindle formation, activation of Cdc25C, actin ring formation, centrosome maturation, and activation of the anaphase-promoting complex. To investigate its functions in mammalian cells further, we used the recently developed small interfering RNA technique specifically to deplete Plk1 in cultured cells. We find that Plk1 depletion results in elevated Cdc2 protein kinase activity and thus attenuates cell-cycle progression. About 45% of cells treated with Plk1 small interfering RNA show the formation of a dumbbell-like DNA organization, suggesting that sister chromatids are not completely separated. About 15% of these cells do complete anaphase but do not complete cytokinesis. Finally, Plk1 depletion significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells. These data provide direct evidence that Plk is required for multiple mitotic processes in mammalian cells and their significance is discussed.

PMID: 12077309

Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20338-43.

Oncogenic H-Ras V12 promotes anchorage-independent cytokinesis in human fibroblasts.
Thullberg M, Gad A, Le Guyader S, Strömblad S.

Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden. minna.thullberg@ki.se

Cell anchorage is required for cell proliferation of untransformed cells, whereas anchorage-independent growth can be induced by oncogenes and is a hallmark of transformation. Whereas anchorage-dependent control of the progression of the G(1) phase of the cell cycle has been extensively studied, it is less clear whether and how anchorage may control other cell cycle phases and whether oncogenes may affect such controls. Here, we found that lack of cell anchorage did not influence progression through the cell cycle S phase, G(2) phase, or most of mitosis of primary human fibroblasts. However, unanchored fibroblasts could not complete cytokinesis. The cleavage furrow and central spindle were still formed in the absence of anchorage, but cells were unable to complete ingression, causing binucleation. Importantly, V12 H-Ras-transformed fibroblasts and two cancer cell lines progressed through the entire cell cycle without anchorage, including through cytokinesis. This indicates that oncogenic signaling may contribute to anchorage-independent growth and tumorigenesis by promoting the final cleavage furrow ingression during cytokinesis.

PMID: 18077377

In the paper above, different methods including: “For cell synchronization, cells were first arrested in G0/G1 by confluence as in ref. 35 and released by replating. For synchronization at the G1/S transition, 5 µM Aphidicolin (Sigma–Aldrich) was then added. Cells synchronized in promethaphase were obtained either by mitotic shake-off (NHDF) or by 2- to 4-h treatment with 40 ng/ml Nocodazole (Sigma–Aldrich), followed by shake-off (BJT and derivatives). Cells were collected, washed, and allowed to adhere to serum-coated tissue-culture dishes or kept in suspension as described in ref. 4.”

For the shake-off method, a very detailed/complicated protocol can be found from this paper:

Cell Biol Int. 2007 Oct;31(10):1184-90.
Ser-10 phosphorylated histone H3 is involved in cytokinesis as a chromosomal passenger.
Song L, Li D, Liu R, Zhou H, Chen J, Huang X.

Department of Biochemistry and Molecular Biology, NanKai University, Tianjin 300071, China.

Ser-10 phosphorylation of histone H3 is revealed to be relative to chromosome condensation at prophase during mitosis. In this report, we demonstrate using immunofluorescence microscopy that the subcellular distribution of the Ser-10 phosphorylated histone H3 was similar to that characteristic of chromosomal passenger proteins during the terminal stages of cytokinesis. Co-immunoprecipitation indicates that the Ser-10 phosphorylated histone H3 is associated with the aurora B, and both of the proteins were compacted into a complex with special ternary structure located in the centre of the midbody. When the level of the Ser-10 phosphorylated histone H3 was reduced by RNA interference, the cells formed an aberrant midbody and could not complete cytokinesis successfully. This evidence suggests that Ser-10 phosphorylated histone H3 is a chromosomal passenger protein and plays a crucial role in cytokinesis.

PMID: 17521927

“Synchronization of the cell cycle was achieved using a double-thymidine block combined with nocodazole treatment. MCF-7 cells in the exponential growth phase were exposed to 2.5 mM thymidine in DMEM/10% FBS for 15 h and then incubated in fresh medium for 9 h. Cells were once again exposed to 2.5 mM thymidine for 15 h and were cultured in fresh DMEM containing 10% FBS. After 5 h, nocodazole was added at a final concentration 80 ng/ml. After 5 h of nocodazole treatment, mitotic cells were purified using the shake-off procedure and were resuspended in fresh DMEM without nocodazole at a concentration of 2–3 × 10e6 cells/ml. Then the cells were maintained at 37 °C for 35 min and allowed to progress into telophase. The cells were collected by centrifugation and subjected to midbody isolation according to the procedure described by Mullins and Mcintosh, 1982 and Sellitto and Kuriyama (1988).”

In the paper below, some methods described for Xenopus cells.

Microsc Res Tech. 1999 Apr 1;45(1):31-42.
Cell cycle analysis and synchronization of the Xenopus laevis XL2 cell line: study of the kinesin related protein XlEg5.
Uzbekov R, Prigent C, Arlot-Bonnemains Y.

Laboratoire de Biologie et Génétique du Développement, Groupe Cycle Cellulaire, CNRS UPR 41, Faculté de Médecine de Rennes, France.

Cell free extracts prepared from Xenopus eggs are one of the most powerful in vitro systems to analyze cell cycle-regulated mechanisms such as DNA replication, nuclear assembly, chromosome condensation, or spindle formation. Xenopus embryos can complete several synchronous cell cycles in the absence of transcription, consequently Xenopus extracts are very helpful to study the molecular level of cellular mechanisms. Many key cell cycle regulators like p34cdc2 and cdk2 have been discovered and characterized using those extracts, but their regulation during somatic cell cycles have only been studied in mammalian cultured cells. In this paper, we describe optimized conditions to obtain cell cycle arrested Xenopus XL2 cultured cells. Synchronization of XL2 cells at different stages of the cell cycle was achieved by serum starvation and drug treatments such as aphidicolin, nocodazole, and ALLN. The degree of synchronization was assessed by indirect fluorescence microscopy and FACS analysis. This method was used to study the cell cycle expression of the Xenopus kinesin-related protein, XlEg5, a microtubule-based motor protein involved in movement and cell division in early development. We found that the expression of the protein was maximum in mitosis and minimum in G1, which correlated with the expression of its messenger RNA. XL2 cultured cells were also used to analyze the ultrastructural sub-cellular localization of XlEg5. During mitosis, the protein was found around the centrosome in prophase, on the spindle microtubules in metaphase, and, interestingly, around the minus end of the midbody microtubules in telophase.

PMID: 10206152

“A classic way for synchronizing cells is to deprive them of growth factors, usually by exposing them to a low concentration of serum. Other techniques involve treatment with compounds which block cells in a specific phase of the cell cycle. For example, inhibitors of DNA synthesis such as hydroxyurea, and methotrexate, do not directly inhibit DNA polymerization but rather block the precursor pathway. These compounds synchronize cells at the G1–S boundary by inhibiting DNA synthesis, whereas drugs such as colcemid, nocodazole, taxol, or ALLN block mitosis. Some of these drugs are sufficiently nontoxic to be of interest for cancer therapy as well as useful for synchronizing cells in culture. If inhibition is specific for one stage of the cell cycle, cells in other phases move forward until they became arrested at the blocked point.”

• G1 phase. “… incubating cells in medium without serum allowed us to obtain cells which were mostly in G1 phase. Adding back serum to the cells released the cell cycle block. The resulting proliferating cell population lost synchrony while traversing G1 phase; thus, it was not possible to collect cells in S phase.”
• S phase. “Aphidicolin prevents DNA chain elongation by inhibiting DNA polymerase alpha… In conclusion, a double treatment including serum deprivation and aphidicolin prevents cells from entering in S phase.”
• G2 phase. “Synchronization of XL2 cells in G2 phase was similar to the synchronization of cells in S phase except that the time for recovery was prolonged. To enrich cells in
  G2 phase, a combination of serum deprivation and aphidicolin block was followed by incubation in full
  medium for 11 h.”
• M phase. “Synchronization of cells in M was achieved with a double block with aphidicolin and nocodazole. Cells cultured in serum-free medium were treated with nocodazole and subsequently when released from this drug they were cultivated for 8 h in serum-free medium in order to get the highest percentage of cells in G2. Sixty percent of all cells were blocked in M phase by adding 0.5 mg/ml of nocodazole to the complete medium.” “Combining serum deprivation, aphidicolin treatment, and release for 8 h, we incubated the cells with ALLN. Almost 57% of the cells were in M phase. Upon removing the ALLN from the culture medium, cells were cultivated in complete medium and polyploidy were not observed among G1 cells.”

“The variation of the percentages observed during the synchronization could be due to differential sensitivity of the cells to the different drugs.”

Other drugs are also available, such as Staurosporine described here.