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Reversine

Manufactured by Selleck Chemicals
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Reversine is a small molecule compound used in laboratory research. It functions as a reversible inhibitor of adenosine monophosphate-activated protein kinase (AMPK) and DNA methyltransferase (DNMT). Reversine can be utilized as a tool compound to study cellular pathways and processes.

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Lab products found in correlation

Colcemid, by Thermo Fisher Scientific (3 mentions) Hesperadin, by Selleck Chemicals (3 mentions) Camptothecin, by Selleck Chemicals (2 mentions) Dmem glutamax, by Thermo Fisher Scientific (2 mentions) Atm inhibitor ku55933, by Bio-Techne (2 mentions) Taxol, by AG Scientific (2 mentions) Vinblastine, by AG Scientific (2 mentions) Dimethylenastron, by AG Scientific (2 mentions) Xtt substrate, by Thermo Fisher Scientific (1 mentions) S trityl l cysteine, by Merck Group (1 mentions) Aphidicolin, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hydroxyurea, by Merck Group (1 mentions) Taxol, by Merck Group (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Mycoalert, by Lonza (1 mentions) Jnk in 8, by Selleck Chemicals (1 mentions)

7 protocols using reversine

1

XTT Cell Viability Assay for Reversine

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XTT cell viability assays were performed by first seeding 4000 LM2-4 cells into a 96-well tissue culture plate. After cells attached overnight the indicated concentrations of Reversine (Selleckchem) or vehicle (DMSO) were then added to the wells in 100 μL phenol-free complete DMEM medium. After 48 h, XTT substrate (Thermo Fisher Scientific) was added to the wells and incubated for 2 h before reading the A450 nm on a plate reader.
Baba S.A., Sun Q., Mugisha S., Labhsetwar S., Klemke R, & Desgrosellier J.S. (2023). Breast cancer stem cells tolerate chromosomal instability during tumor progression via c-Jun/AXL stress signaling. Heliyon, 9(9), e20182.
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2

Synthesis and Storage Protocols for Cancer Inhibitors

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The TTK inhibitors NTRC 0066-0 [18 (link)], MPI-0479605 [20 (link)] and Mps-Bay2b [21 (link)] were synthesized according to published protocols. Reversine was purchased from Selleck Chemicals; S-trityl-L-cysteine (STLC) from Sigma Aldrich. The source of all other anti-cancer agents is provided in Supplementary Table 2 of Uitdehaag et al. [41 (link)]. All compounds were stored as powders and freshly dissolved in dimethyl sulfoxide (DMSO) as 10 mM stocks.
Libouban M.A., de Roos J.A., Uitdehaag J.C., Willemsen-Seegers N., Mainardi S., Dylus J., de Man J., Tops B., Meijerink J.P., Storchová Z., Buijsman R.C., Medema R.H, & Zaman G.J. (2017). Stable aneuploid tumors cells are more sensitive to TTK inhibition than chromosomally unstable cell lines. Oncotarget, 8(24), 38309-38325.
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3

Cell Line Characterization and Maintenance

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HT1080 6TG cells were a kind gift from Eric Stanbridge (University of California, Irvine) and Saos-2 were provided by Roger Reddel (CMRI). IMR90 cells were purchased from ATCC and IMR90 E6E7 derived in the Karlseder laboratory. Phoenix cells were purchased from ATCC. Identity of all cell lines were verified by Cell Bank Australia using short tandem repeat profiling. HT1080 6TG, HeLa, Saos-2 and derivatives were cultured at 37 °C, 10% CO2, and atmospheric oxygen, in DMEM (Life Technologies) supplemented with 1% non-essential amino acids (Life Technologies), 1% Glutamax (Life Technologies), and 10% bovine growth serum (Hyclone). IMR90 and derivatives were cultured at 37 °C, 10% CO2, and 3% O2, in DMEM, supplemented with 1% non-essential amino acids, 1% Glutamax, 10% fetal bovine serum (Life Technologies). The following compounds were used in cell treatments: dimethyl sulfoxide (DMSO, Sigma-Aldrich), colcemid (Life Technologies), Taxol (Sigma), reversine (Selleck Chemicals), KU-55933 (Calbiochem), Hesperadin (Selleck chemicals), Aphidicolin (Sigma-Aldrich) and Hydroxyurea (Sigma-Aldrich). All cell lines were tested for mycoplasma contamination (MycoAlert, LT07-118, Lonza) and were found to be negative.
Masamsetti V.P., Low R.R., Mak K.S., O’Connor A., Riffkin C.D., Lamm N., Crabbe L., Karlseder J., Huang D.C., Hayashi M.T, & Cesare A.J. (2019). Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection. Nature Communications, 10, 4224.
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4

Fibroblast and Cancer Cell Culture Protocols

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Human IMR-90 primary lung fibroblasts (ATCC) and their derivatives were grown in Glutamax-DMEM (Gibco) supplemented with 0.1 mM nonessential Amino Acids and 15% Fetal Bovine Serum. HT1080 6TG cells were grown in Glutamax-DMEM supplemented with 0.1 mM Nonessential Amino Acids and 10% Bovine Growth Serum. All cells were grown at 7.5% CO2 and 3% O2. Colcemid (Life Technologies), Taxol (A. G. Scientific), vinblastine (A. G. Scientific), dimethylenastron (A. G. Scientific), hesperadin (Selleck), reversine (Selleck) and camptothecin (Selleck) were used at indicated concentration. ATM inhibitor (KU-55933) (Tocris) was used at 10 μM. FACS analysis was performed as described27 . Cells were tested for mycoplasma contamination and found negative.
Hayashi M.T., Cesare A.J., Rivera T, & Karlseder J. (2015). Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection. Nature, 522(7557), 492-496.
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5

Fibroblast and Cancer Cell Culture Protocols

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Human IMR-90 primary lung fibroblasts (ATCC) and their derivatives were grown in Glutamax-DMEM (Gibco) supplemented with 0.1 mM nonessential Amino Acids and 15% Fetal Bovine Serum. HT1080 6TG cells were grown in Glutamax-DMEM supplemented with 0.1 mM Nonessential Amino Acids and 10% Bovine Growth Serum. All cells were grown at 7.5% CO2 and 3% O2. Colcemid (Life Technologies), Taxol (A. G. Scientific), vinblastine (A. G. Scientific), dimethylenastron (A. G. Scientific), hesperadin (Selleck), reversine (Selleck) and camptothecin (Selleck) were used at indicated concentration. ATM inhibitor (KU-55933) (Tocris) was used at 10 μM. FACS analysis was performed as described27 . Cells were tested for mycoplasma contamination and found negative.
Hayashi M.T., Cesare A.J., Rivera T, & Karlseder J. (2015). Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection. Nature, 522(7557), 492-496.
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6

Tumorsphere Formation Assay in Methylcellulose

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Primary tumorsphere formation in methylcellulose was assessed in cells grown under anchorage-independent conditions. LM2-4 cells (10,000) were cultured in 0.9 mL of 1% methylcellulose/complete DMEM medium in ultra-low adhesion 24-well dishes (Corning, Corning, NY, USA) and cells cultured for 8–10 days. Cells were treated with vehicle (DMSO) or the indicated doses of SP600125, JNK–IN–8, or Reversine (all from Selleckchem) concurrent with embedding in methylcellulose. Primary tumorspheres were assessed by counting colonies consisting of at least 6 cells from 4 fields per well with a 10x objective.
Baba S.A., Sun Q., Mugisha S., Labhsetwar S., Klemke R, & Desgrosellier J.S. (2023). Breast cancer stem cells tolerate chromosomal instability during tumor progression via c-Jun/AXL stress signaling. Heliyon, 9(9), e20182.
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7

Reversine, HDAC, and EZH2 Inhibitor Treatments

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Cells were treated with reversine (Cayman Chemical Company) at a concentration of 0.5 µM for either 24 h (for MDA-MB-231) or 48 h (4T1). The HDAC inhibitor vorinostat (Sigma-Aldrich; also known as SAHA) was used at a concentration of 0.5 µM for 24 h, and the methyltransferase inhibitor GSK-126 (XcessBio; EZH2) was used at a concentration of 5 µM for 5 days. Long-term reversine treatment was done 24 h after cells were passaged, and the medium was replaced with normal cell medium after 48 h. GSK923295 (Selleck Chemicals) was used at the concentration of 50 nM for 24 h. DMSO was used as the vehicle control.
Agustinus A.S., Al-Rawi D., Dameracharla B., Raviram R., Jones B.S., Stransky S., Scipioni L., Luebeck J., Di Bona M., Norkunaite D., Myers R.M., Duran M., Choi S., Weigelt B., Yomtoubian S., McPherson A., Toufektchan E., Keuper K., Mischel P.S., Mittal V., Shah S.P., Maciejowski J., Storchova Z., Gratton E., Ly P., Landau D., Bakhoum M.F., Koche R.P., Sidoli S., Bafna V., David Y, & Bakhoum S.F. (2023). Epigenetic dysregulation from chromosomal transit in micronuclei. Nature, 619(7968), 176-183.
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