Synchronization of HeLa cells was either done by a single 2 mM thymidine (Sigma-Aldrich, T1895) arrest for 24 h (Figs 1 , 2a,b , 3d , 4a,c , 5c–e,g and 6 and Supplementary Figs 1c, 2b, 3C–E, 4C–D, 5A ) or a double-thymidine arrest (22 h arrest followed after 9 h by a 17 h arrest (Figs 2d , 3a–c and 5a,f and Supplementary Figs 3a, 4a and 5b ). After washout drugs were added: 1 μM Nocodazole (M1404, Sigma-Aldrich), 0.1 μM BI2536 (S1109, Selleck), 10 μM QVD (SML0063, Sigma-Aldrich), 0.5 μM reversine (BML-SC104, Enzo Life Sciences), 2.5 μM (in Fig. 7 ) or 1 μM (in all other Figures) ABT-737 (S1002, Selleck) were added. Dimethyl sulfoxide (D5879, Sigma-Aldrich) was used as solvent control. G2 time points were harvested after 8 h and the first mitotic time point (‘M') 11 h after release from thymidine arrest. All mitotic time points were harvested by shake-off. Doxycycline (D9891, Sigma-Aldrich) was used in the indicated concentrations. Staurosporine (S-9300, LC Laboratories) was used on asynchronous cells for 4 h at 1 μM.
T1895
Manufactured by Merck Group
Sourced in United States
T1895 is a laboratory equipment product. It is designed for laboratory use and its core function is to perform specific tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
M1404, by Merck Group (4 mentions)
Thymidine, by Merck Group (3 mentions)
Nocodazole, by Merck Group (2 mentions)
Bi2536, by Selleck Chemicals (2 mentions)
Snap cell block, by New England Biolabs (2 mentions)
Thymidine, by New England Biolabs (2 mentions)
Snap cell substrates, by New England Biolabs (2 mentions)
Snap cell block s9106s, by Thermo Fisher Scientific (2 mentions)
647 sir s9102s, by Thermo Fisher Scientific (2 mentions)
Tmr star s9105s, by Thermo Fisher Scientific (2 mentions)
Snap cell 430 s9109s, by Thermo Fisher Scientific (2 mentions)
Oregon green s9104s, by Thermo Fisher Scientific (2 mentions)
P35g 1.5 14 c, by MatTek (2 mentions)
D8668, by Merck Group (2 mentions)
D0776, by Merck Group (2 mentions)
D0901, by Merck Group (2 mentions)
Doxycycline, by Merck Group (1 mentions)
D5879, by Merck Group (1 mentions)
Abt 737, by Selleck Chemicals (1 mentions)
Staurosporine, by LC Laboratories (1 mentions)
22 protocols using t1895
1
HeLa Cell Synchronization and Drug Treatment
2
C2C12 Cell Cycle Arrest Conditions
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The mouse myoblast cell line C2C12 was maintained in a growth state in Dulbecco's modified Eagle's medium (DMEM) (Gibco by Thermo Fisher Scientific) containing 1 g l−1 glucose, 1% l -glutamine and 1% penicillin/streptomycin supplemented with 20% fetal bovine serum (FBS). NIH3T3 cells were maintained in DMEM containing 4.5 g l−1 glucose, 1% l -glutamine and 1% penicillin/streptomycin supplemented with 20% FBS. Cells were grown in a 5% CO2 cell culture incubator at 37°C. Differentiation stimuli were induced by exchanging media with 2% HS instead of 20% FBS.
C2C12 myoblasts were arrested in S phase by thymidine (Sigma-Aldrich, T1895) and in G2 phase by Cdk1 inhibitor (RO-3306, Sigma-Aldrich, SML0569) for 48 h. A single treatment of thymidine was applied, while additional RO-3306 was added at intervals of 8–12 h.
C2C12 myoblasts were arrested in S phase by thymidine (Sigma-Aldrich, T1895) and in G2 phase by Cdk1 inhibitor (RO-3306, Sigma-Aldrich, SML0569) for 48 h. A single treatment of thymidine was applied, while additional RO-3306 was added at intervals of 8–12 h.
3
Synchronized cell culture for RNAi
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Human cervical cancer cells (HeLa), BJ human fibroblasts and human osteosarcoma cells (U2OS) were purchased from ATCC repository. Hela 1.3 cells are a gift from Titia de Lange27 (link). BJ EHLT (hTERT immortalized, SV40 LT antigen transformed) were obtained by retroviral transduction of BJ cells with hTERT (Addgene plasmid #1773) and Large T SV40 antigen (Addgene plasmid # 21826). Cells were maintained in Dulbecco Modified Eagle Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics. In synchronization experiments, cells were seeded at 40% confluence and exposed to 2 mM thymidine (T-1895, SIGMA) for 16 h (I block), followed by 8 h release in fresh medium, and again exposed to 2 mM thymidine for additional 16 h (II block). Then cells were released in fresh medium and collected by trypsinization at different time points for further analysis. RNAi was performed by transfecting cells 2 days before synchronization at 20% confluence with 5 nM siRNA (scrambled sequence, two different sequences against PARP1, WRN and TRF1: PARP1 siRNA Origene SR300098B/C, TRF1 siRNA Origene SR322000B/C, SCR siRNA Origene SR30004, WRN siRNA ORIGENE SR322215B/C and POLYPLUS INTERFERIN #409-10 as Transfection reagent).
4
Synchronizing Cells and Labeling Mitochondria
5
Cell Cycle Synchronization and Immunoprecipitation
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For immunoprecipitation, MCF7 cells and 293T cells were treated with thymidine (2 mm , #T1895; Sigma‐Aldrich, St. Louis, MO, USA) for 18 h and released into fresh medium for 10 h and then blocked with 2 mm thymidine again for 18 h. After that, the cells were released into fresh medium for 2 h and treated with nocodazole (0.1 μg·mL−1, #M1404; Sigma‐Aldrich) for 10 h. Then the mitotic cells were collected for immunoprecipitation.
For analysis of flow cytometry and western blot, SUM159PT cells, MCF7 cells, and 293T cells were treated with nocodazole (0.1 μg·mL−1, #M1404; Sigma‐Aldrich) for 10 h and released into nocodazole‐free medium; then the cells were collected at the indicated time for further analysis.
For analysis of flow cytometry and western blot, SUM159PT cells, MCF7 cells, and 293T cells were treated with nocodazole (0.1 μg·mL−1, #M1404; Sigma‐Aldrich) for 10 h and released into nocodazole‐free medium; then the cells were collected at the indicated time for further analysis.
6
Culturing murine embryonic stem cells
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R1 mESCs, ZHBTc4 (mESC lines) (Niwa et al., 2000 (link)) and pre-iPS cells were cultured on gelatin-coated tissue culture plates in knockout DMEM (Thermo Fisher, 10829018) supplemented with 15% fetal calf serum (Lonsera, S711-004S/NN02953), 1× nonessential amino acids (Gibco, 11140050), 2 mmol/L L-glutamine (Gibco, 35050061), 1% (v/v) nucleoside mix (Sigma-Aldrich, A-4036, T-1895, C-4654, G-6264, U-3003), 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich, M6250), 1,000 U/mL recombinant leukemia inhibitory factor (LIF) (Millipore, ESG1107).
7
Nucleotide Supplementation for SARS-CoV-2 Infection
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Following exposure to SARS-CoV-2 (0.1 MOI) for 1 h, Huh7 were washed once with PBS 1× and then incubated with fresh DMEM containing 2% of heat-inactivated FBS supplemented with 5 μM deoxy-adenosine (dA), deoxy-cytidine (dC) and deoxy-guanidine (dG)90 (link) (D8668, D0776 and D0901, Sigma) for 48 h before being collected for downstream analyses. dN supplementation in Calu-3 was carried out by incubating cells with medium containing 50 μM dA and dC, 5 μM dG and deoxy-thymidine (dT, T1895, Sigma) for 24 h.
8
Synchronizing HCT-116-RAD21-AID Cells for Experimentation
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HCT-116-RAD21-AID cells were obtained from Natsume et al.39 (link). The cells were cultured in McCoy’s 5A medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. Cells were selected with 100 μg/ml G418 and 100 μg/ml HygroGold prior to experiments.
To avoid heterogeneity due to cell cycle, HCT-116-RAD21-AID cells were synchronized at the G1/S transition. First, to arrest cells in the S-phase, cells were grown in medium supplemented with 2 mM thymidine (Sigma-Aldrich T1895) for 12 hours. Cells were then resuspended in medium and allowed to grow for 12 hours to exit S-phase. To arrest at the G1/S transition, we grew cells in medium supplemented 400 μM mimosine (Sigma-Aldrich M025) for 12 hours. Lastly, we replaced medium with either 400 μM mimosine + 500 μM indole-3-acetic acid (auxin; Sigma-Aldrich I5148)-supplemented medium to degrade RAD21 or 400 μM mimosine-supplemented medium alone as an untreated control; cells were incubated with or without auxin for 6 hours then harvested for experiments. Synchronization was confirmed by IF (Extended Data Fig. 2a ).
To avoid heterogeneity due to cell cycle, HCT-116-RAD21-AID cells were synchronized at the G1/S transition. First, to arrest cells in the S-phase, cells were grown in medium supplemented with 2 mM thymidine (Sigma-Aldrich T1895) for 12 hours. Cells were then resuspended in medium and allowed to grow for 12 hours to exit S-phase. To arrest at the G1/S transition, we grew cells in medium supplemented 400 μM mimosine (Sigma-Aldrich M025) for 12 hours. Lastly, we replaced medium with either 400 μM mimosine + 500 μM indole-3-acetic acid (auxin; Sigma-Aldrich I5148)-supplemented medium to degrade RAD21 or 400 μM mimosine-supplemented medium alone as an untreated control; cells were incubated with or without auxin for 6 hours then harvested for experiments. Synchronization was confirmed by IF (
9
NG108-15 Cells Exosome Exposure
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NG108-15 cells (HB-12317 ATCC) were cultured in DMEM with 10% FBS and supplemented with 0.1 mM hypoxanthines, 400 nM aminopterin, and 16 μM thymidine (H9377, A1784, and T1895 Sigma-Aldrich). NG108-15 cells were plated on a 60 mm cell culture dish or a 35 mm confocal dish in DMEM and cultured in 10% exosome-free FBS. After plating, 4 types of SC exosomes (L-EXO and H-EXO; MC-EXO and miR-21-EXO; 15/5 μg resuspended in PBS) were added daily for 3 days.
10
Synchronizing Cells and Labeling Mitochondria
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Cells were synchronised with a double thymidine block21 (link)
. After incubation in 2 mM thymidine (Sigma-Aldrich, T1895) for 19h, PBS washes, collection using trypsin-EDTA (Thermo Fisher Scientific, 15400054), and centrifugation, cells were treated with Snap-Cell substrates to label old mitochondria, washed, and plated on cell culture dishes, or poly-L-lysine (Merck Millipore, A-005-C, final concentration 1 μg/ml) coated 35 mm glass bottom dishes (MatTek, P35G-1.5-14-C). After 10 hours, thymidine was added for 17 hours, cells washed and returned to MEGM. Cells divided 21-26h after the second thymidine release 21 (link)
.
Snap-Cell Block or Snap-Cell substrates (New England Biolabs) were applied for 30min at 37°C as indicated, diluted in full MEGM with 0.5% BSA (Thermo Fisher Scientific, 15260037) at 5-10μM (Snap-Cell Block S9106S), 1.5-3μM (Snap-Cell 647-SiR S9102S), 3μM (Snap-Cell TMR-Star S9105S), 5μM (Snap-Cell 430 S9109S), 5μM (Snap-Cell Oregon Green S9104S).
. After incubation in 2 mM thymidine (Sigma-Aldrich, T1895) for 19h, PBS washes, collection using trypsin-EDTA (Thermo Fisher Scientific, 15400054), and centrifugation, cells were treated with Snap-Cell substrates to label old mitochondria, washed, and plated on cell culture dishes, or poly-L-lysine (Merck Millipore, A-005-C, final concentration 1 μg/ml) coated 35 mm glass bottom dishes (MatTek, P35G-1.5-14-C). After 10 hours, thymidine was added for 17 hours, cells washed and returned to MEGM. Cells divided 21-26h after the second thymidine release 21 (link)
.
Snap-Cell Block or Snap-Cell substrates (New England Biolabs) were applied for 30min at 37°C as indicated, diluted in full MEGM with 0.5% BSA (Thermo Fisher Scientific, 15260037) at 5-10μM (Snap-Cell Block S9106S), 1.5-3μM (Snap-Cell 647-SiR S9102S), 3μM (Snap-Cell TMR-Star S9105S), 5μM (Snap-Cell 430 S9109S), 5μM (Snap-Cell Oregon Green S9104S).
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