What is double thymidine block?
Double thymidine block High concentrations of thymidine interrupt the deoxynucleotide metabolism pathway through competitive inhibition, thus blocking DNA replication. A single treatment with thymidine arrests cells throughout S phase, so a double treatment acts to induce a more uniform block in early S phase.
How do I sync my cell cycle?
Wash your cells in serum-free media. Re-suspend or seed them at the right concentration. Seed and incubate for anywhere between 24 and 72 hours. Check for synchronization in G1 using DNA dyes in flow cytometry, as mentioned later in this article.
Why do cells synchronize?
Synchronization of cells is essential to study cell cycle specific events. If, for example, one suspects that a given DNA repair pathway is used in a particular cell cycle phase, the protocol can be used to enrich cells in each phase of the cell cycle and analyze the cellular response to DNA damage.
Which compound is used for cell synchrony at S phase?
The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M.
What is thymidine block?
Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block.
How do you make thymidine solution?
To prepare a 100 mM stock of thymidine solution, dissolve 2.42g thymidine in 90 ml tissue culture grade H2O. Adjust final volume to 100 ml. Sterilize by filtration.
What is the function of thymidine?
Thymidine kinases have a key function in the synthesis of DNA and therefore in cell division, as they are part of the unique reaction chain to introduce thymidine into the DNA. Thymidine is present in the body fluids as a result of degradation of DNA from food and from dead cells.
What is thymidine used for?
The thymidine analog 5-bromo-2′-deoxyuridine (BrdU) is used to label dividing cells. BrdU is injected intraperitoneally and incorporates into DNA being replicated where it will remain for the life of the cell.
How is cell synchronization by double thymidine block used?
Cell Synchronization by Double Thymidine Block Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication.
How does thymidine block arrest cell at G1 / s boundary?
Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points.
How are thymidine analogs used in cell culture?
Modified analogs. Bromodeoxyuridine (BrdU) is another thymidine analog that is often used for the detection of proliferating cells in living tissues. 5-Ethynyl-2´-deoxyuridine (EdU) is a thymidine analog which is incorporated into the DNA of dividing cells and is used to assay DNA synthesis in cell culture or living tissues.
Which is DNA nucleoside pairs with thymidine?
Thymidine. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DNA. In cell biology it is used to synchronize the cells in G1/early S phase. Before the boom in thymidine use caused by the need for thymidine in the production of the antiretroviral drug azidothymidine (AZT),…