Phleomycin

Manufactured by Merck Group

Sourced in United States

Phleomycin is a laboratory reagent used for selective cell culture and molecular biology applications. It is an antibiotic compound that inhibits the growth of various microorganisms, including bacteria and fungi.

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30 protocols using phleomycin

Phleomycin-Induced DNA Damage Response

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Phleomycin (Sigma) was added at 30 μg ml⁻¹ for 2 h. Cells were processed for analysis 30 min after Phleomycin treatment unless indicated otherwise. After a 30 min recovery in ESC medium, the cells were collected and processed for the following experiments. For detection of the DNA damage response in the extended period, the cells were given 6 h to recover after Phleomycin treatment and were processed for H2AX immunostaining. In the DNA fragmentation assay, the cells were given 15 h to recover. To check the mutagenesis potential, the cells were treated with 30 μg ml⁻¹ Phleomycin for 2 h and cultured for one passage after each treatment. Cells were irradiated at 10 Gy, allowed to recover for 2 h, and then lysates were collected for immunoblot analysis. For the H₂O₂ treatment (TBHP; stable chemical form of H₂O₂) cells were treated with 350 μM for 30 min in PBS.

Skamagki M., Correia C., Yeung P., Baslan T., Beck S., Zhang C., Ross C.A., Dang L., Liu Z., Giunta S., Chang T.P., Wang J., Ananthanarayanan A., Bohndorf M., Bosbach B., Adjaye J., Funabiki H., Kim J., Lowe S., Collins J.J., Lu C.W., Li H., Zhao R, & Kim K. (2017). ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors. Nature cell biology, 19(9), 1037-1048.

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Phleomycin-Induced DNA Damage Kinetics

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Cells were seeded (4 × 10⁴ cells/well) in a 24-well plate with 12mm circle cover glassed (Thermo Fisher Scientific) in 1 ml medium per well and cultured for 24 h. Then the cells were treated with phleomycin (Sigma-Aldrich) (30 μM/ml) for 0 min, 15 min, 30 min, 1 h, 2 h, or 4 h. The cells were then washed with PBS twice, fixed with 4% formaldehyde in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS solution for 10 min at room temperature. After washing with PBS 3 times, the cells were incubated with blocking buffer (PBS containing 10% goat serum, 1% BSA and 0.1% tween) for 1 h at room temperature. The cells were then incubated in phospho-H2AX or phospho-DNA-PKCs primer antibody (Abcam, Cambridge, MA) (5 μg/ml in PBS containing 1% BSA and 0.1% tween) overnight at 4°C, and the fluorochrome-conjugated second antibody (5 μg/ml in PBS containing 1% BSA and 0.1% tween) for 1 h at room temperature, followed by rinsing in PBS 3 times for 5 min each. The slides were mounted with a small drop of ProLong^® Gold Antifade Mountant with DAPI (Thermo Scientific, Pittsburgh, PA) and viewed under a fluorescence microscope.

Wang W., Lu Y., Stemmer P.M., Zhang X., Bi Y., Yi Z, & Chen F. (2015). The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair. Oncotarget, 6(29), 28269-28281.

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Dictyostelium Cell Survival Assay

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Exponentially growing Dictyostelium cells were collected, resuspended to a density of 1 × 10⁶ cells/ml in HL5, and separated into 1 ml aliquots. Cells were exposed to the indicated concentrations of DNA damaging agent phleomycin (Sigma) or mock-treated and incubated at 100 rpm for 1 h whilst shaking. Afterwards, cells were diluted to 1 × 10⁴ cells/ml in KK2 and replicates of 250 cells were plated on 140 mm SM agar plates in association with K. aerogenes. Survival was assessed by observing Dictyostelium plaque formation after 3–7 days.

Kolb A.L., Gunn A.R, & Lakin N.D. (2017). Redundancy between nucleases required for homologous recombination promotes PARP inhibitor resistance in the eukaryotic model organism Dictyostelium. Nucleic Acids Research, 45(17), 10056-10067.

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Yeast Cell Culture Protocol

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Strains are listed in Table 1. Cells were grown in yeast extract plus supplements medium (YES). Stock solutions of caffeine (Sigma Aldrich AB, Stockholm, Sweden) (100 mM) were prepared in water stored at −20°C. HU (Sigma Aldrich AB) was dissolved in water at a concentration of 1 M and stored at −20°C. Phleomycin (Sigma Aldrich AB) was dissolved in water and stock solutions (10 μg ml⁻¹) stored at −20°C. MBC (Carbendazim/methylbenzimidazol-2yl carbamate) and latrunculin B (Lat B) (Sigma Aldrich AB) were stored at −20°C as 10 mg ml⁻¹ stock solutions in DMSO.

Alao J.P., Sjölander J.J., Baar J., Özbaki-Yagan N., Kakoschky B, & Sunnerhagen P. (2014). Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override. Molecular Microbiology, 92(4), 777-796.

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Visualizing T. brucei Organelle Dynamics

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Px I–II^−/− BS T. brucei and polyclonal rabbit antibodies against Px were generated previously [19] (link). Bovine holo-transferrin, 10,000 Da dextran conjugated to Alexa Fluor 488, LysoTracker Green DND-26, DIDS, and MitoTracker Red CMXRos were purchased from Life Technologies. FeCl₃, deferoxamine mesylate, bovine Hb, bovine apo-transferrin, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), DAPI, and phleomycin were from Sigma. FCS was from Biochrome. Affinity-purified bovine transferrin antibodies were purchased from Bethyl Laboratories Inc. The monoclonal mouse anti-p67 antibody was a kind gift of Dr. James D. Bangs, Buffalo and the polyclonal rabbit anti-aldolase antibody was kindly provided by Dr. Christine Clayton, Heidelberg.

Hiller C., Nissen A., Benítez D., Comini M.A, & Krauth-Siegel R.L. (2014). Cytosolic Peroxidases Protect the Lysosome of Bloodstream African Trypanosomes from Iron-Mediated Membrane Damage. PLoS Pathogens, 10(4), e1004075.

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Inhibitors for DNA Damage Response

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The PARP inhibitors ABT888 (Selleck Chemicals) and olaparib (Selleck Chemicals) were used at a final concentration of 1–10 μM. PARG (PDD00017273; Sigma), ATM (KU-55933; Selleck Chemicals), ATR (AZ-20; Tocris), and DNA-PK (NU-7441; Selleck Chemicals) inhibitors were used at a final concentration of 10 μM. The HDAC inhibitors TSA (Sigma), SAHA (Abcam), and Romidepsin (Selleck Chemicals) were used at a final concentration of 0.2, 5, and 5 µM, respectively. Hydroxyurea (Sigma, H8627-100G) was used at final concentration of 1 mM and phleomycin (Sigma) was used at final concentration of 500 µM.

Rother M.B., Pellegrino S., Smith R., Gatti M., Meisenberg C., Wiegant W.W., Luijsterburg M.S., Imhof R., Downs J.A., Vertegaal A.C., Huet S., Altmeyer M, & van Attikum H. (2020). CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks. Nature Communications, 11, 5775.

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Cell Competition Assay with XRCC4 and KU70

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For Xrcc4 mutant cell competition experiments, 1.6 x 10⁵ cells were co-transfected in suspension with 0.45 μg empty vector and either 50 ng empty vector or 50 ng GFP-expression plasmid using Lipofectamine 2000 (Invitrogen). For KU70 complementation cell competition experiments, 1.6 x 10⁵ cells were co-transfected in suspension with 0.35 μg empty vector, 0.15 μg empty vector or hKU70-expression plasmid, and either 50 ng empty vector or 50 ng GFP-expression plasmid using Lipofectamine 2000 (Invitrogen). 18 hours after transfection, cells were counted, mixed 5:1, uncolored vs. GFP⁺ marked cells, and 5 x 10⁴ cells plated in triplicate. 6 hours after cell plating growth medium was replaced with media containing phleomycin (Sigma-aldrich, P9564). After two days incubation, GFP⁺ frequencies were scored on a Beckman Coulter CytoFlex LX. Fold enrichment of cultures transiently co-transfected with GFP-expression plasmid normalized to 0 μg/mL phleomycin control. Plots represent the mean of triplicate samples from three independent experiments (n = 3).

Willis N.A., Panday A., Duffey E.E, & Scully R. (2018). Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier. PLoS Genetics, 14(7), e1007486.

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Transfection of Leishmania Promastigotes

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Transfections were performed in an Amaxa Nucleofector (Lonza) using program U-033 as described previously [47] (link). Briefly, 5 × 10⁷ logarithmic phase promastigotes were suspended in Human T Cell Nucleofector (Lonza) and transfected with 2–10 μg DNA. Parasites were allowed to recover in 10 mL of culture medium without selection drug for 24 h before centrifugation and plating on medium M199 agar plates containing 10% (v/v) iFBS, 50 U/mL penicillin, 50 mg/mL streptomycin, 0.1 mM adenine, 0.023 mM hemin, 25 mM HEPES sodium salt, pH 7.4 (all from Sigma), and the selective drug(s). Hygromycin (Invitrogen) was used at 15 μg/mL, G418 (Sigma) at 15 μg/mL, and phleomycin (Sigma) at 17.5 μg/mL. Colonies were picked after 1–2 weeks and transferred to liquid medium.

Specht S., Liedgens L., Duarte M., Stiegler A., Wirth U., Eberhardt M., Tomás A., Hell K, & Deponte M. (2017). A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast. Redox Biology, 15, 363-374.

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Cell Transfection and Small Molecule Treatments

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JetPEI (PolyPlus), Fugene6 (Promega), Lipofectamine 2000 (Invitrogen), INTERFERin (PolyPlus), Neocarzinostatin (NCS, Sigma) Phleomycin (Sigma), XAV-939 (Tocris), Mimosine (Sigma), RO-3306 (Millipore), ATMi KU55933 (Tocris).

Nagy Z., Kalousi A., Furst A., Koch M., Fischer B, & Soutoglou E. (2016). Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs. PLoS Genetics, 12(2), e1005791.

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Yeast Growth Sensitivity Assays

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Yeast strains were grown at 30°C to saturation and diluted to an OD₆₀₀ of 0.5 prior to spotting 5-fold serial dilutions on plates at 30°C or 34°C, as indicated, for 2–4 days. Plates contained the indicated concentrations of caffeine (Sigma-Aldrich), rapamycin (Roche), phleomycin (Sigma-Aldrich), 6-azauracil (6AU) (Roche), or 5-fluoorotic (5-FOA) (RPI). For the Bur1 growth assay, BUR1 deletion shuffle strains were grown on media lacking uracil to maintain the wild-type BUR1 plasmid before plating on media containing the 5-FOA. All experiments were performed at least three times.

DiFiore J.V., Ptacek T.S., Wang Y., Li B., Simon J.M, & Strahl B.D. (2020). Unique and Shared Roles for Histone H3K36 Methylation States in Transcription Regulation Functions. Cell reports, 31(10), 107751.

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