Nocodazole

Manufactured by Selleck Chemicals

Sourced in United States

Nocodazole is a laboratory reagent used to disrupt microtubule formation in cells. It functions by binding to tubulin, preventing its polymerization into microtubules. This cell cycle arrest property makes nocodazole a valuable tool in cell biology research.

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39 protocols using nocodazole

Cell Cycle Synchronization with Nocodazole

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Nocodazole was purchased from Selleck (#S2775) and dissolved in DMSO at the concentration of 2 nM. Before the experiment, K562 cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum, 1% Penicillin-streptomycin and 1 nM Nocodazole for 20 h. For flow cytometry analysis, the medium containing Nocodazole was removed, and the cells were re-suspended in 400 μL cold 1× PBS and 1100 μL cold fixing solution (100% ethyl alcohol) and stored overnight at 4 °C. The next day cells were centrifuged to remove the fixing solution, washed three times with 1× PBS and re-suspended in 500 μL 1× PBS. RNase A (ThermoFisher, 20 mg L⁻¹) was introduced to remove the interference of RNA at 37 °C for 1 h. Then the nuclear DNAs of K562 cells were stained by PI (ThermoFisher, 50 mg L⁻¹) in a dark place at 4 °C for 1 h. One million K562 cells in total were detected by flow cytometry, with the obvious fluorescence peak corresponding to 2n/4n DNAs, which represented the cell cycle position. For Paired-seq, after removing the Nocodazole, K562 cells were washed three times and re-suspended in DMEM supplemented with 0.2% F68 and 4% FBS.

Zhang M., Zou Y., Xu X., Zhang X., Gao M., Song J., Huang P., Chen Q., Zhu Z., Lin W., Zare R.N, & Yang C. (2020). Highly parallel and efficient single cell mRNA sequencing with paired picoliter chambers. Nature Communications, 11, 2118.

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Cell Cycle Synchronization Protocols

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For double thymidine block, PC-3 cells were treated with 2 mM thymidine (Selleck, catalog no. S4803) for 18 hours and were released into regular medium for 9 hours, after which the cells were further blocked by treatment with thymidine for another 15 hours. For thymidine-nocodazole block, PC-3 cells were treated with 2 mM thymidine for 24 hours and were released into regular medium for 3 hours, after which cells were further blocked by treatment with nocodazole (Selleck, catalog no. S2775) for another 12 hours. At the indicated time points after release, the cells were harvested for Western blot analysis using previously described methods.

Ma J., Ren D., Wang Z., Li W., Li L., Liu T., Ye Q., Lei Y., Jian Y., Ma B., Fan Y., Liu J., Gao Y., Jin X., Huang H, & Li L. (2024). CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity. Science Advances, 10(21), eadk8908.

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Cell Cycle Synchronization Protocols

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3D4/21 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and then cultured for 24 h in serum-free supplemented medium (cells arrested in the G0/G1 phase). 3D4/21 cells were seeded in 6-well plates until the cell density was about 80% following treatment with thymidine (2 mmol/L, Sigma, St. Louis, MO, USA), nocodazole (80 ng/mL, Selleckchem, Houston, TX, USA) for 24 h (cells arrested in the S, G2/M phase, respectively).

Zhu M., Li X., Sun R., Shi P., Cao A., Zhang L., Guo Y, & Huang J. (2021). The C/EBPβ-Dependent Induction of TFDP2 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Proliferation. Virologica Sinica, 36(6), 1341-1351.

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Selective CDK2 Inhibition and Mitochondrial Dynamics

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Two μmol/L CVT‐313 (Selleck), an effective ATP‐competitive selective CDK2 inhibitor, was incubated for 12 h and used for UMUC3 and T24. Mdivi‐1 (Selleck), a mitochondrial division inhibitor 1, is a selective transmembrane inhibitor of mitochondrial division that inhibits DRP1 and dynamin I (Dnm1). Five μmol/L Mdivi‐1 was incubated with the cell cultures for 24 h. Mito‐Tracker Red (Beyotime) was used at a concentration of 200 μmol/L for 15 min to stain the mitochondria. Nocodazole (Selleck) was applied at a dosage of 100 nmol/L for 12 h to induce the synchronization of cells. Then, the synchronized cells were released in a fresh medium.

Liu X., Liu S., Piao C., Zhang Z., Zhang X., Jiang Y, & Kong C. (2021). Non‐metabolic function of MTHFD2 activates CDK2 in bladder cancer. Cancer Science, 112(12), 4909-4919.

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Cell Culture and Drug Treatment Protocol

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The 293T and HeLa cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, USA) supplemented with 10% fetal bovine serum (ExCell Bio, Shanghai, China), 100 U/ml streptomycin and 100 U/ml penicillin. Cells were cultured at 37°C in a humidified incubator under 5% CO₂. For drug treatment, cells were treat with Paclitaxel or nocodazole (S1150, S2775; Selleckchem, Houston, USA) for 18 hours.

Jing R., Xi J., Leng Y., Chen W., Wang G., Jia W., Kang J, & Zhu S. (2017). Motifs in the amino-terminus of CENP-A are required for its accumulation within the nucleus and at the centromere. Oncotarget, 8(25), 40654-40667.

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Synchronizing Osteosarcoma and Kidney Cells

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Osteosarcoma U2OS and embryonic kidney HEK 293T cells were cultured in DMEM supplemented with 10% FBS and 2 mM l-glutamine at 37°C in a humidified atmosphere of 5% CO₂. U2OS and 293T cells were synchronized to G₂ phase by incubation with 10 µM RO-3306 (217699; Calbiochem) and to prometaphase with 100 ng/ml nocodazole (M1404; Sigma-Aldrich) or 10 µM monastrol (M8515; Sigma-Aldrich) for 16 h. To collect mitotic U2OS cells, cells arrested at prometaphase were harvested using the mechanical shake-off procedure. Metaphase arrest was achieved by incubation with 100 ng/ml nocodazole for 16 h, followed by incubation in nocodazole-free medium with 20 µM MG132 for 90 min (474790; Calbiochem). To inhibit PLK1, 100 nM BI2536 (S1109; Selleck Chemicals) was applied to cells treated with nocodazole or MG132 for 90 min.

Zhao W., Liu J., Zhang X, & Deng L.W. (2016). MLL5 maintains spindle bipolarity by preventing aberrant cytosolic aggregation of PLK1. The Journal of Cell Biology, 212(7), 829-843.

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Mutagenic Analysis of MST2 Signaling

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Flag-MST2 has been described [19 (link)]. Point mutations were generated by the QuikChange Site-Directed PCR Mutagenesis Kit (Stratagene) and verified by sequencing. HEK293T, HEK293GP, and HeLa cell lines were purchased from American Type Culture Collection (ATCC) and cultured as ATCC instructed. Attractene (Qiagen) was used for transient overexpression of proteins in HEK293T and HEK293GP cells following the manufacturer’s instructions. SiRNA oligos were purchased from Dharmacon (the target sequences were: siMST2-1: CCACAAGCACGATGAGTGA; siMST2-2: GCCCATATGTTGTAAAGTA; siMST2-3: GAACTTTGGTCCGATGATT) and transfected with HiPerfect reagent from Qiagen (at the final concentration of 40 nM). Transient transfections were done with Attractene reagent (Qiagen) following the manufacturer’s instructions. Nocodazole (100 ng/ml for 16 h) and Taxol (100 nM for 16 h) (Selleck Chemicals) were used to arrest cells in G2/M phase. VX680 (Aurora-A, -B, -C inhibitor), BI2536 (PLK1 inhibitor), Purvalanol A (CDK1/2/5 inhibitor), SB216763 (GSK-3 inhibitor) and MK2206 (Akt inhibitor) were also from Selleck Chemicals. RO3306 (CDK1 inhibitor) was from ENZO Life Sciences. Kinase inhibitors for MEK-ERK (U0126) and p38 (SB203580) were from LC Laboratory. All other chemicals were either from Sigma or Thermo Fisher.

Chen X., Chen Y, & Dong J. (2016). MST2 Phosphorylation at Serine 385 in Mitosis Inhibits its Tumor Suppressing Activity. Cellular signalling, 28(12), 1826-1832.

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Intracerebroventricular Injection in Rats

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A guide cannula (Plastics One, Roanoke, VA) was implanted in rats for intracerebroventricular microinjection [54 (link)]. The coordinates were 1.5 mm lateral, 1.0 mm posterior from bregma, and 4.0 mm in depth. The cannula location was confirmed histologically at the end of experiment. Paclitaxel and nocodazole (Selleck Chemicals) were dissolved in 5% of 2-hydroxypropyl-β-cyclodextrin (Sigma) for injection. Intracerebroventricular injection (5 μL/injection at 0.5uL/min) was made using a microsyringe and the needle was held for 1 min before retraction.

You Z., Zhang S., Shen S., Yang J., Ding W., Yang L., Lim G., Doheny J.T., Tate S., Chen L, & Mao J. (2018). Cognitive Impairment in a rat model of neuropathic pain: Role of Hippocampal Microtubule Stability. Pain, 159(8), 1518-1528.

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Compound Dosage Optimization for In Vitro Studies

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Bortezomib, MLN2238, nocodazole and RO-3306 were purchased from SelleckChem (Houston, TX) for the in vitro studies. Compounds were resuspended in DMSO and frozen down in 20 microliter aliquots to limit freeze-thaw cycles. Compounds were added as noted in the figure legends. In vitro studies used 15 nM for Bortezomib and 100 nM for MLN2238 or are otherwise specified in the text. For the pulse experiments, we used 2.5 micromolar of MLN2238.

Hong A.L., Tseng Y.Y., Wala J.A., Kim W.J., Kynnap B.D., Doshi M.B., Kugener G., Sandoval G.J., Howard T.P., Li J., Yang X., Tillgren M., Ghandi M., Sayeed A., Deasy R., Ward A., McSteen B., Labella K.M., Keskula P., Tracy A., Connor C., Clinton C.M., Church A.J., Crompton B.D., Janeway K.A., Van Hare B., Sandak D., Gjoerup O., Bandopadhayay P., Clemons P.A., Schreiber S.L., Root D.E., Gokhale P.C., Chi S.N., Mullen E.A., Roberts C.W., Kadoch C., Beroukhim R., Ligon K.L., Boehm J.S, & Hahn W.C. (2019). Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition. eLife, 8, e44161.

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Cell Cycle Synchronization and Analysis

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Mino and Jeko-1 cells were synchronized to G2/M phase by treatment with nocodazole (Selleckchem). To synchronize cells in G1, the synchronized G2/M cells were washed with PBS and grown in fresh medium for 12 h. Briefly, Mino and Jeko-1 cells were collected 24 h after transfection, and treated with 50 ng/ml nocodazole for 24 h, then released in fresh medium. Cells were collected at 12 and 18 h after release and processed to ethanol fixing (70% ethanol, ice-cold), RNase A-pretreating (0.5 mg/ml at 37 °C for 30 min) and propidium iodide staining (50 μg/ml). Cell-cycle progression was measured by flow cytometry.

Wang X., Sehgal L., Jain N., Khashab T., Mathur R, & Samaniego F. (2016). LncRNA MALAT1 promotes development of mantle cell lymphoma by associating with EZH2. Journal of Translational Medicine, 14, 346.

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