Before cell synchronization, it is important to ensure that SFFs are maintained at <70% confluence. Synchronization of cells in the G1 and G2/M phases were achieved by treatment with 40 μM lovastatin (MCE) and 200 ng/mL nocodazole (MCE) for 17 h, respectively. Synchronization of cells in S phase required two sequential treatments: cells were first treated with 2 μg/mL aphidicolin (Abcam) for 17 h, then released in aphidicolin-free medium for 4.5 h and again treated with a second dose of drugs for 17 h. DMSO was used as a control. After cell synchronization, cell-cycle distributions were analyzed by flow cytometry as described above.
Aphidicolin
Manufactured by Abcam
Sourced in United States
Aphidicolin is a DNA polymerase inhibitor that blocks cell division by inhibiting DNA replication. It is commonly used in cell biology research to synchronize cells in the G1/S phase of the cell cycle.
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Lab products found in correlation
Hydrogen peroxide, by Merck Group (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Ku55933, by Selleck Chemicals (1 mentions)
Ve 821, by Selleck Chemicals (1 mentions)
Thymidine, by Merck Group (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Cytochalasin a, by Merck Group (1 mentions)
S3226, by Merck Group (1 mentions)
Verteporfin, by Bio-Techne (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Colcemid, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Abcam (1 mentions)
5 protocols using aphidicolin
1
Cell Synchronization Protocols for Cell Cycle Analysis
2
DNA Damage Response Modulation Protocol
3
Cell Cycle Synchronization and EdU Labeling
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Cells with and without transfection were grown on glass-bottom dishes and synchronized at the G1/S transition by 2 mM thymidine (Sigma-Aldrich, T1895) for 15 h, followed by culturing in fresh DMEM for 10 h, and treatment with 2 μg/mL aphidicolin (abcam, ab142400) for 15 h.
To incorporate EdU in HeLa cells, the cells were bathed with growing medium containing EdU for 30 min after releasing them from the G1/S transition for 1 h, followed by transfection and growth in fresh DMEM for 2 days. The cy5 dye was then conjugated to EdU using the Click-iT EdU Imaging Kit (Thermo Scientific, C10340) according to the manufacturer’s instructions. Imaging data were acquired using the spinning disk confocal system described above.
To incorporate EdU in HeLa cells, the cells were bathed with growing medium containing EdU for 30 min after releasing them from the G1/S transition for 1 h, followed by transfection and growth in fresh DMEM for 2 days. The cy5 dye was then conjugated to EdU using the Click-iT EdU Imaging Kit (Thermo Scientific, C10340) according to the manufacturer’s instructions. Imaging data were acquired using the spinning disk confocal system described above.
4
Preparation of Hypertonic and Hypotonic Solutions
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Sucrose(Macklin, S818046) was dissolved in ddH 2 O, the concentration of the stock solution was 1 mol/L. The stock solution is diluted by KSOM (260 mOsm) into a hypertonic solution(340 mOsm) of sucrose with a final concentration of 100 mM. We used ddH 2 O to dilute KSOM into a 70% hypotonic solution (170 mOsm). (-)-Blebbistatin (Sigma,B0560) was dissolved in DMSO at a concentration of 25 mM. The stock solution was diluted by KSOM to a working concentration of 1 μM. Dissolve LPA(Cayman,62215) in ethonal at the stock concentration of 1 mM. The stock solution was diluted by KSOM to a working concentration of 5 μM. Cytochalasin A(Sigma,C6637) was dissolved in DMSO at the stock concentration of 0.5 mg/mL. The stock solution was diluted by KSOM to a working concentration of 1 μg/mL. Dissolved S3226 (Sigma,SML 1996) in ddH 2 O to prepare a stock solution with a concentration of 2 mg/mL. The stock solution was diluted by KSOM to a working concentration of 20 nM. Dissolved Verteporfin(Tocris,5305) in DMSO to prepare a stock solution with a concentration of 10 mg/mL. The stock solution was diluted by KSOM to a working concentration of 2 μg/mL. Dissolved Aphidicolin (Abcam,ab142400) in DMSO to prepare a stock solution with a concentration of 10 mg/mL. The stock solution was diluted by KSOM to a working concentration of 2 μg/mL.
5
Synchronizing NIH-3T3 Cells for Cell Cycle Analysis
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Mouse embryonic fibroblast NIH-3T3 cells were cultured in DMEM (GIBCO, Carlsbad, CA, USA) supplemented with 10% FBS (GIBCO, Carlsbad, CA, USA) and 1% Pen/Strep (GIBCO, Carlsbad, CA, USA) at 37℃, 5% CO 2 .
To obtain G1/S synchronized NIH-3T3 cells, cells were cultured with starvation treatment (DMEM with 0.5% FBS and 1% Pen/Strep) for 48 h, followed with fresh medium containing 1% Aphidicolin (Abcam, Cambridge, MA, USA) for 18 h before collection. With this procedure, about 97% of the collected cells were G1/S cells based on FACS (Fluorescence-Activated Cell Sorting) analysis (Supplementary Figure S1A, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168). To obtain mitotic cells, cells in culture reached 70-80% confluence were treated with colcemid (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for 12 h, and mitotic cells were shaken-off and collected for mitotic chromosome purification. The purity of the collected mitotic cells was about 77% by FACS analysis (supplementary Figure S1B, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168).
To obtain G1/S synchronized NIH-3T3 cells, cells were cultured with starvation treatment (DMEM with 0.5% FBS and 1% Pen/Strep) for 48 h, followed with fresh medium containing 1% Aphidicolin (Abcam, Cambridge, MA, USA) for 18 h before collection. With this procedure, about 97% of the collected cells were G1/S cells based on FACS (Fluorescence-Activated Cell Sorting) analysis (Supplementary Figure S1A, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168). To obtain mitotic cells, cells in culture reached 70-80% confluence were treated with colcemid (100 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) for 12 h, and mitotic cells were shaken-off and collected for mitotic chromosome purification. The purity of the collected mitotic cells was about 77% by FACS analysis (supplementary Figure S1B, http://www.biosciencetrends.com/action/ getSupplementalData.php?ID=168).
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