UTA6 and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and antibiotics, penicillin and streptomycin. Cells were plated onto plastic dishes for large-scale cell culture and glass-bottomed dishes (LabTek) or 13 mm round coverslips for imaging. For inhibition studies, cells were treated with 10 µM MG132 (1748, TOCRIS). Double-thymidine block for synchronisation was performed on 60% confluent cell cultures by treating cells with 2 mM thymidine (ACROS organics) for 24 h, releasing cells from the thymidine treatment for 12 h, treating cells with a second round of 2 mM thymidine for 12 h and finally releasing them for thymidine treatment for 9–10 h for mitotic cell enrichment. For large-scale prometaphase cell enrichment, soon after the second round of thymidine release, 5 µM DMA was added (Enzo Life Sciences) and 14 h later rounded up cells were collected by shake-off. For anaphase cell enrichment, prometaphase cells were washed with warm DMEM and released into drug free medium for 45 min.
Thymidine
Manufactured by Thermo Fisher Scientific
Sourced in United States
Thymidine is a nucleoside that is a component of DNA. It serves as a key building block for the synthesis and replication of DNA molecules in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Thymidine, by Merck Group (12 mentions)
Nocodazole, by Merck Group (11 mentions)
Hypoxanthine, by Thermo Fisher Scientific (10 mentions)
Aminopterin, by Thermo Fisher Scientific (8 mentions)
Imject alum, by Thermo Fisher Scientific (7 mentions)
Rpmi medium, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Peg1500, by Roche (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Polyethylene glycol 1500, by Roche (3 mentions)
Mg132, by Merck Group (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
Mg132, by Bio-Techne (2 mentions)
Penicillin, by GoldBio (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Thymidine, by Chem-Impex (2 mentions)
50 protocols using thymidine
1
Synchronized Cell Culture and Enrichment
2
HeLa Cell Synchronization and Analysis
3
Repair Pathway Inhibition Assays
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For different repair pathway inhibition assays, cells were pre-treated with B02 (HY-101462, 20 μM; MCE), mirin (HY-117693, 25 μM; MCE), Nu7026 (HY-15719, 2 μM; MCE), or Olaparib (HY-10162, 10 nM; MCE) for 12 h. For the DTB cell-cycle arrest experiment, cells were pre-treated with thymidine (T9250-1G, 2 mM; Sigma) for 18 h, thymidine was removed, the cells were cultured in normal media without thymidine for 9 h and then, thymidine was re-added to the cells for a second round of 18 h. Inhibitor treated cells were transfected using the Neon Transfection System (Invitrogen) as described above. Transfected cells were treated with the inhibitors for another 2 d before cultured in normal media.
4
Synchronizing HeLa Cells for Transfection
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HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, USA) at 37 ℃ with 5% CO2.
1.0 μg of plasmid pmGFP and 0.9 μg of plasmid pCAGGS were transfected into HeLa cells (5 × 105 cells) using Lipofectamine 2000 (Life Technologies, USA).
After incubation for 24 h, cell synchronization using the double thymidine block method was applied to arrest cells in the early S phase. thymidine (Sigma-Aldrich, USA) was added to the medium upto a final concentration of 2 mM and incubated for 14 h at 37 °C with 5% CO2. The cells were washed twice with 2 mL of PBS. Then, 2 mL of medium was added and incubated for 9 h at 37 °C with 5% CO2. Following incubation, thymidine was added to the medium upto a final concentration of 2 mM and incubated for 14 h at 37 °C with 5% CO2. Finally, the medium was replaced with 2 mL of Opti-MEM I (Thermo Fisher Scientific, USA) with thymidine (final concentration of 2 mM).
1.0 μg of plasmid pmGFP and 0.9 μg of plasmid pCAGGS were transfected into HeLa cells (5 × 105 cells) using Lipofectamine 2000 (Life Technologies, USA).
After incubation for 24 h, cell synchronization using the double thymidine block method was applied to arrest cells in the early S phase. thymidine (Sigma-Aldrich, USA) was added to the medium upto a final concentration of 2 mM and incubated for 14 h at 37 °C with 5% CO2. The cells were washed twice with 2 mL of PBS. Then, 2 mL of medium was added and incubated for 9 h at 37 °C with 5% CO2. Following incubation, thymidine was added to the medium upto a final concentration of 2 mM and incubated for 14 h at 37 °C with 5% CO2. Finally, the medium was replaced with 2 mL of Opti-MEM I (Thermo Fisher Scientific, USA) with thymidine (final concentration of 2 mM).
5
Cell Cycle Synchronization and Analysis
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HeLa cells were regularly cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin in a humidified incubator supplemented with 5% CO2 at 37°C. Synchronizing cells to different phases referred to established protocols (37 (link)–39 (link)). Generally, G1-phase enriched cells were obtained by treating cells with 400 μM L-mimosine (Sigma) for 20–24 h. To achieve a maximum S-phase population, cells were blocked with 2 mM thymidine (Sigma) for 20 h and released in fresh medium for 10 h, followed by 20 h of blocking with thymidine and finally culturing in fresh medium for another 4 h. Cultures containing G2-phase arrested cells were obtained by incubating with fresh medium containing 0.1 μg/ml KaryoMAX® Colcemid (Life Technologies) for 6 h after two rounds of thymidine blocking. Flow cytometry was used to assess cell cycle distribution. Cells were fixed with 70% ethanol and then stained with 20 μg/ml propidium iodide (PI) solution containing 0.1% Triton X-100 and 200 μg/ml RNase A, referring to standard protocol (40 (link)). Cell cycle distribution, through quantifying the PI fluorescence intensity, was determined in a FC500 MPL system (Beckman Coulter). At least 15 000 cells were collected for each condition and the generated cell cycle histograms were analyzed with Watson pragmatic model in Flowjo software package.
6
Cell Cycle Synchronization Protocol
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Cells were arrested in mitosis with a thymidine-nocodazole block, and cell synchrony was monitored by flow cytometry of propidium iodide-stained cells. Cell cycle synchronization was adapted from the protocol of Whitfield et al [81 (link)]. Briefly, cells were blocked for 24 hours with 2 mM thymidine (Fisher Scientific), released for 3 hours by washing out the thymidine and adding fresh DMEM/F12 medium with 1% FBS and 1% AA. Then the cells were blocked with 100 ng/ml nocodazole (Sigma-Aldrich) for 12 hours to arrest all cells in mitosis. The cells were released from nocodazole block into G1 synchronously by washing out nocodazole and adding fresh medium. The cells were harvested at 0, 2, 4, and 8 hours after release, washed with PBS, and fixed overnight in 70% ethanol. Fixed cells were then centrifuged, washed, resuspended in PBS containing RNase A (100 μg/mL) and propidium iodide (50 μg/mL), and incubated for 20 minutes on ice and in the dark. Stained cells were analyzed using BD LSR II flow cytometer (BD Biosciences) and cell cycle population distribution was determined by ModFit LT software (Verity software house, Topsham, ME, USA).
7
Characterization of GnT-I and FUT8 Knockout CHO Cells
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The CHO cell line lacking endogenous GnT-I, Pro-5WgaRI3C (Lec1) (Stanley and Chaney 1985 (link)), was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The FUT8-knockout CHO cell line was established in our laboratory (Yamane-Ohnuki et al. 2004 (link)). Both CHO cell lines were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10% (v/v) dialyzed fetal bovine serum, 0.1 mmol/L hypoxanthine and 16 mmol/L thymidine (all from Invitrogen, Carlsbad, CA).
8
Culturing Endothelial Cells for Glycocalyx Research
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Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were purchased from Lonza (Slough, UK) and grown in medium 199 (Invitrogen, Paisley, UK) supplemented with foetal bovine serum (10%), -endothelial cell growth factor (1 ng/ml), bovine neural extract ( ), thymidine ( ), heparin (10 U/ml), penicillin (100 U/ml) and streptomycin ( ). All supplements were purchased form Sigma. Before experiment, endothelial cells were collected by incubating with trypsin-EDTA solution (0.5%, Sigma, Dorset, UK) for 5 min and then plated on a glass, non-coating coverslip at a density of (HUVECs) and (HAECs). They were further cultured for a week, allowing full recovery of the glycocalyx on the cell surface (here defined as an intensive, continuous FITC-WGA binding layer) (Bai and Wang 2012 (link)).
9
Enrichment and Treatment of Mitotic HeLa Cells
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HEK293T and HeLa cells were routinely maintained in advanced Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen) with 10% (v/v) fetal bovine serum (FBS; HyClone) and 100 units/ml of penicillin plus 100 μg/ml streptomycin (Gibco). To enrich mitotic cells, HeLa cells were treated with 2.5 mM thymidine (Invitrogen) for 14 h and then released into a fresh DMEM. For monastrol (50 μM, Sigma) treatment, the drug was added into the culture medium 8 h after release and maintained for 2 h. Metaphase HeLa cells for quantification of chromosome misalignment were enriched with MG132 (20 μM, Sigma) treatment. For immunoprecipitation of GFP-tagged proteins, HeLa cells were treated with nocodazole (0.1 ng/μl) for 14 h and then harvested for experiments. For Hesperadin (100 nM, Sigma) treatment, the drug was added into the culture medium and maintained for 30 min.
10
Synchronizing HeLa cells for immunofluorescence and immunopurification
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