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| Product Name | Cell Cycle Synchronization Kit, Double Thymidine Block Method |
| Catalog No. | CCAT-HMM-0066 |
| Description | Cell cycle synchronization kit employing the classic double thymidine block method to reversibly arrest adherent and suspension cultured cells at the G1/S phase boundary. The kit provides sterile-filtered thymidine solution (200 mM) and optimized protocols for single and double block procedures. The first thymidine block arrests cells throughout S-phase by depleting dCTP pools through allosteric inhibition of ribonucleotide reductase. After release into thymidine-free medium for 8-9 hours (approximately one S-phase duration), a second thymidine block captures all cells at the G1/S transition as they re-enter S-phase simultaneously. Release yields a highly synchronized cell population (>85% in S-phase within 2 hours). |
| Intended Use | Reversible cell cycle synchronization at G1/S boundary for studying cell cycle-dependent processes, DNA replication timing, DNA damage response, drug sensitivity profiling, and coordinated biochemical analysis of cell cycle regulatory proteins. |
| Principle / Technology | Thymidine excess feedback inhibits ribonucleotide reductase, blocking dCTP synthesis and halting DNA replication; cells accumulate at G1/S boundary; removal of thymidine releases the block, allowing synchronous entry into S-phase; double block increases synchronization efficiency. |
| Detection Method | Add thymidine to 2 mM final concentration; incubate 14-18 hours (first block); wash cells 3× with PBS; culture in complete medium 8-9 hours; add thymidine to 2 mM; incubate 14-16 hours (second block); wash and release into complete medium; collect cells at time points for cell cycle analysis. |
| Sample Type | Adherent and suspension cultured mammalian cells with doubling time 18-30 hours; HeLa, HEK293, U2OS, NIH/3T3, CHO cells. |
| Performance Range / Specifications | Synchronization efficiency: 80-95% of cells arrested at G1/S boundary after double block; synchrony maintained for 4-6 hours post-release; S-phase peak at 2-4 hours after release. |
| Sensitivity / LOD | Cell cycle progression detectable at 30-minute intervals post-release by PI flow cytometry; synchronization efficiency verified by G1 DNA content peak (>85% of events). |
| Specificity | Thymidine specifically inhibits ribonucleotide reductase; does not affect RNA or protein synthesis; fully reversible upon washout; minimal cytotoxicity with optimized exposure time. |
| Reaction Conditions / Protocol | See protocol steps above; key parameters: thymidine concentration (2 mM), block duration (14-18 hours), release interval (8-9 hours), second block (14-16 hours). |
| Components / Formulation | Thymidine (200 mM, sterile-filtered, 10 mL), detailed protocol with troubleshooting guide. |
| Storage Conditions | Store at -20 °C in single-use aliquots; avoid repeated freeze-thaw cycles. |
| Shelf Life | 12 months from date of manufacture at -20 °C. |
| Package Specifications | 10 mL (sufficient for ~100 synchronization experiments in T25 flasks or 6-well plates). |
| Product Form | Sterile-filtered colorless liquid solution. |
| Quality Control | Each lot tested for sterility, endotoxin <0.1 EU/mL, thymidine concentration verification by absorbance at 267 nm (ε = 9,650 M-1cm-1), and synchronization efficiency in HeLa cells (>85% G1/S arrest). |
| Key Features | Classic double thymidine block method; sterile-filtered and ready-to-use; reversible synchronization; non-toxic and gentle on cells; validated for multiple cell lines; complete protocol with troubleshooting included. |
| Purity | Thymidine ≥99% purity by HPLC; cell culture grade; endotoxin tested. |
| Concentration | 200 mM stock solution; use at 2 mM final concentration (1:100 dilution). |
| Activity / Unit Definition | Ribonucleotide reductase inhibition effective at 2 mM thymidine in standard culture medium. |
| Molecular Weight | Thymidine: 242.23 g/mol (C10H14N2O5). |
| Source / Origin | Synthetic thymidine from pharmaceutical-grade nucleoside synthesis. |
| pH Range / Optimal pH | Thymidine stock solution pH 7.0-7.4. |
| Shipping Conditions | Cold pack recommended; ambient acceptable for short transit. |
| Expiration Date / Stability | 12 months at -20 °C; thawed aliquots stable for 1 week at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with DMEM, RPMI-1640, MEM, and most standard cell culture media. Synchronization efficiency varies by cell line doubling time; optimal for cells with 18-30 hour cycle. Not recommended for primary cells with limited proliferative capacity. |
| Recommended Buffer System | Thymidine dissolved in sterile PBS, pH 7.4. |
| Application Notes / Precautions | Optimize release interval based on your cell line's S-phase duration. Verify synchronization by flow cytometry before each experiment. Serum deprivation can enhance synchronization when combined with thymidine block for some cell types. Check cell morphology after double block — some rounding is normal, but >20% floating cells indicates excessive toxicity; reduce block duration. Include asynchronous control in all experiments for comparison. |
| Batch-to-Batch Consistency | Thymidine concentration within ±5% of specification; endotoxin <0.1 EU/mL; synchronization efficiency verified (>85% G1/S arrest in HeLa cells). |
For research use only, not for clinical use.
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