Cell Cycle Synchronization

Holmes, Paul; Al‐Rubeai, Mohamed

doi:10.1002/0471250570.spi035

Cited by 3 publications

(5 citation statements)

References 22 publications

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“…Our flow cytometric analysis showed that highly synchronous cell populations could be obtained by this method; 86% of cells were in G1 phase 4 h after incubation of the shake‐off cells. Although the decay of synchrony inevitably occurs with time, due to the temporal variability of individual cell in the cell cycle (Doyle et al, 1993; Holmes and Al‐Rubea, 2000), a high proportion of synchronous cells were still obtained at 8 h in this study (70% of cells in S phase). Obviously, since G2/M phases in CNE‐2Z cells last for approximately 100 min, compared to a S‐phase duration of 9 h and G1‐phase duration of 6 h, and because the temporal variability of individual cell in the cell cycle, it is difficult to obtain a very high proportion of cells in G2/M phase.…”

Section: Discussionmentioning

confidence: 56%

“…Ideally, the synchronization method should not perturb the metabolic pathways of the cells. Thus physical methods are generally preferable to chemical methods and the technique of mitotic shake‐off is the method of choice (Doyle et al, 1993; Holmes and Al‐Rubea, 2000). Our flow cytometric analysis showed that highly synchronous cell populations could be obtained by this method; 86% of cells were in G1 phase 4 h after incubation of the shake‐off cells.…”

Section: Discussionmentioning

confidence: 99%

“…Cell preparation and analysis followed the methods described by Holmes and Al‐Rubea (2000). Cells were fixed in 70% ethanol at −20°C for 30 min and stored at this temperature until required for analysis.…”

Section: Methodsmentioning

confidence: 99%

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Regulatory volume decrease is actively modulated during the cell cycle

Wang

Chen

Zhu

et al. 2002

Journal Cellular Physiology

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Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 microM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress.

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Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

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Regulatory volume decrease is actively modulated during the cell cycle

Wang

Chen

Zhu

et al. 2002

Journal Cellular Physiology

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“…Gentle agitation of the growth medium will thus remove the mitotic cells, which can then be used as a starting point for a synchronous population. This method can provide highly synchronous cultures of mitotic cells (10,12,13). CNE-2Z cells were incubated at a density of 1.5 ϫ 10 4 cells/cm 2 in culture medium in 175-cm 2 plastic tissue culture flasks at 37°C using a humidified atmosphere of 5% CO 2 for 24 h. After cell attachment and before the mitotic cells were harvested, the cultures were washed to remove unattached dead cells.…”

Section: Cell Synchronizationmentioning

confidence: 99%

“…Cell preparation and analysis followed the methods described by Holmes and Al-Rubeai (13). Cells were fixed in 70% ethanol at Ϫ20°C for 30 min and stored at this temperature until required for analysis.…”

Section: Cell Cycle Analysismentioning

confidence: 99%

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Chen

Wang

Zhu

et al. 2002

American Journal of Physiology-Cell Physiology

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Patch-clamping and cell image analysis techniques were used to study the expression of the volume-activated Cl− current, I Cl(vol), and regulatory volume decrease (RVD) capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated a Cl− current with a linear conductance, negligible time-dependent inactivation, and a reversal potential close to the Cl− equilibrium potential. The sequence of anion permeability was I− > Br− > Cl− > gluconate. The Cl− channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and ATP inhibited I Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by a double chemical-block (thymidine and hydroxyurea) technique. The expression of I Cl(vol) was cell cycle dependent, being high in G1 phase, downregulated in S phase, but increasing again in M phase. Hypotonic solution activated RVD, which was cell cycle dependent and inhibited by the Cl− channel blockers NPPB, tamoxifen, and ATP. The expression of I Cl(vol) was closely correlated with the RVD capacity in the cell cycle, suggesting a functional relationship. Inhibition of I Cl(vol) by NPPB (100 μM) arrested cells in G0/G1. The data also suggest that expression of I Cl(vol) and RVD capacity are actively modulated during the cell cycle. The volume-activated Cl− current associated with RVD may therefore play an important role during the cell cycle progress.

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