Epithelial OC cell lines (A2780, A2780_CR5, SKOV3, HEYC2, OV90, IOSE, IGROV, OVMUNA, OV90) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) as described previously [50 (link)]. Cisplatin-resistant A2780_CR5 was established by continuous exposure to increasing concentrations of cisplatin [50 (link)]. Cell lines were authenticated in 2012 by ATCC and tested for mycoplasma contamination (Manassas, VA). High-grade serous ovarian tumors (unpaired samples; chemo-naïve, stage 3–4 or recurrent, platinum resistant), and ovarian surface epithelium (OSE) were surgically collected with informed consent from all subjects (IRB approved protocol IUCRO-0280), snap-frozen, and stored in liquid nitrogen [51 (link)]. Cisplatin (CDDP) was purchased from Calbiochem (Billerica, MA), mitomycin C (MMC) was purchased from Sigma Chemical Co. (St. Louis MO), and H2O2 was purchased from EMD Millipore (Billerica, MA). NF-κB inhibitor Bay-11–7082 was purchased from Santa Cruz Biotech (Santa Cruz, CA). LZRS-HOTAIR was a gift from Dr. Howard Chang (Stanford University; Addgene plasmid # 26110). Full-length HOTAIR was cloned into pAV5S vector containing a 98-mer aptamer sequence and as a vector control, aptamer cloned into pAV5S was used to account for any possible RNA-dependent signaling effects [52 (link)].
Mitomycin c mmc
Manufactured by Merck Group
Sourced in United States, Germany
Mitomycin C (MMC) is a laboratory-grade reagent manufactured by Merck Group. It is a DNA-alkylating agent used in various research applications, including the study of cellular processes, DNA damage response, and cancer biology. MMC functions by forming covalent crosslinks between DNA strands, which can lead to cell cycle arrest and cell death. This product is intended for research purposes only and should be handled with appropriate safety precautions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Hydroxyurea hu, by Merck Group (5 mentions)
Cisplatin, by Merck Group (4 mentions)
Subcellular protein fractionation kit, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Nicolet 6700, by Thermo Fisher Scientific (3 mentions)
Bay11 7082, by Santa Cruz Biotechnology (2 mentions)
Cytochalasin b cytb, by Merck Group (2 mentions)
Chromosome medium b, by Harvard Bioscience (2 mentions)
Histone h3 fl 136 and anti vinculin h 10, by Santa Cruz Biotechnology (2 mentions)
Anti ps824kap 1 a300 767a, by Fortis Life Sciences (2 mentions)
Anti ps317γh2ax 05636, by Merck Group (2 mentions)
Anti ps15p53 ab1431 and anti polq ab80906, by Abcam (2 mentions)
Anti brdu 555627, by BD (2 mentions)
Parpi rucaparib ag 014699, by Selleck Chemicals (2 mentions)
Abt 888, by Abbvie (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
72 protocols using mitomycin c mmc
1
Ovarian Cancer Cell Line Maintenance and Characterization
2
Evaluating FANCA Plasmid Transfection
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U2OS cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). U2OS cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin–neomycin (PSN) antibiotic mixture (Gibco) at 37 °C with 5% CO2. To evaluate the transfection efficiency, FANCA plasmids were co-transfected with pEGFP-N2 vector (Clontech) into U2OS cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. In experiments evaluating cell sensitivity to ICL damage, 2 μM mitomycin C (MMC; Sigma) was added into the culture medium 24 h after transfection.
3
Cytogenetic Assay Reagents and Protocols
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Fetal bovine serum (FBS), Ham’s F-10 medium, and Phytohaemaglutinin (PHA) were commercially procured by Gibco (Fisher Scientific, Loughborough, Leicestershire, UK Ltd). Mitomycin C (MMC) and Cytochalasin-B (Cyt-B) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were of the highest grade commercially available. Stocks of the compounds and solutions were stored at 4 °C until use. Treatments were performed and based on the initial stocks of the tested compounds.
4
Mitomycin C-treated MSCs for T-cell co-culture
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BM-MSCs and normal fibroblast cells (WI38) were purchased from Cambrex Bioscience Walkersville (East Rutherford, NJ, USA) and ATCC (Manassas, VA, USA), respectively. Cells were cultured in alpha-MEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S, 100 mg/mL; Gibco-BRL, NY, USA), and 2 mM l -glutamine (Gibco-BRL). PD-MSCs were harvested from the inner side of the chorioamniotic membrane of the placenta as described previously (16 (link)). PD-MSCs and BM-MSCs at passages 6 to 8 were used for assays. The cells were treated with 50 μg/mL of mitomycin C (MMC; Sigma-Aldrich) for 50 min to stop cell division and then used as feeder cells for co-culture with naïve or stimulated T cells isolated from peripheral blood (PB).
5
Cell Viability Assay for Genotoxic Agents
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Cells were seeded into 96 well plates (4000 cells/well) and incubated for 4 hours before treatment with methyl methanesulfonate (MMS) (Sigma-Aldrich, Saint Louis, MO, USA), mitomycin-C (MMC) (Sigma-Aldrich), cisplatin (Hospira) or UVB (Vilber Lourmat, Bio Spectra V5, 312 nm, cells exposed in 100 μL medium, additional 100 μL added after UVB exposure). The drugs were added on day 0, and cells were analyzed 24, 48 and 72 h after UVB treatment and 24, 72 and 96 h after MMC, MMS and cisplatin treatment. MTT (3-(4.5-Dimethylthiazol-2-yl)-2.5 diphenyl-tetrazolium bromide) was added to the cells and OD was measured at 565 nm, and the average from at least 6 wells was used to calculate cell survival.
6
Cell Fractionation and Mitomycin C Exposure
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U2OS and HT1080 cells were obtained from ATCC and maintained as recommended. Cell fractionation was carried out using the Subcellular Protein Fractionation Kit (ThermoFisher Scientific) and as described by the manufacturer. Exposure of cells to 1 μM mitomycin C (MMC; Sigma) occurred in regular growth medium at 37 °C for 24 h.
7
Evaluating DNA Repair Efficiency Using Chromosome Breakage Assay
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Hypersensitivity to the chromosome-breaking effect of crosslinking agents is a reliable marker of homologous recombination (HR) efficiency.11 (link) For POI genes involved in DNA repair and not expressed exclusively in germ cells, chromosomal breakage studies were performed in cultures of peripheral lymphocytes obtained from the patient, the mother when available, and a healthy woman as a control. The experiments were performed at the Gustave Roussy Institute (Villejuif, France), following a standard in-house protocol.7 (link), 8 (link), 9 (link),12 (link) Fresh peripheral blood lymphocytes were cultured under standard conditions for karyotyping. DNA damage was induced by treating the cells with mitomycin C (MMC; Sigma) for 48 h to examine cellular hypersensitivity to DNA crosslinking agents. Three treatment conditions were used: without MMC to analyze spontaneous DNA damage and with 150 or 300 nM MMC as recommended.11 (link) Chromosome breaks were scored by an experienced cytogeneticist using at least 20 metaphases.
8
Recombinant Protein Expression in E. coli and H. volcanii
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The oligonucleotides used in this study were purchased from Takara (Dalian, China). Escherichia coli strain DH5α was used for gene cloning, and the Rosetta2(DE3)pLysS strain was used for recombinant protein expression. The expression vectors pET28a and pDEST17 were used to express recombinant proteins. The H. volcanii strain and plasmids for gene knockout were provided by Dr Allers (University of Nottingham, UK). Pyrococcus furiosus genomic DNA was purchased from the American Type Culture Collection (ATCC). KOD-plus DNA polymerase was purchased from Toyobo. Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad. The crosslinking reagent BS3 and the DNA damage reagents 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), and methyl methanesulfonate (MMS), methyl methanesulfonate (MMS), N3-methyladenine and adenine were purchased from Sigma–Aldrich (St Louis, MO). All other chemicals and reagents were of analytical grade.
9
Cytogenetic Evaluation of Potassium Tetraborate
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The heparinized blood samples were collected from four healthy males aged from 25 to 30 years old, respectively, non-smoking, non-alcoholic, not under drug therapy and with no recent history of exposure to mutagens. Human peripheral blood cultures were set up according to a slight modification of the protocol described by Evans and O’Riordan (1975[7 (link)]). The heparinized blood (0.5 ml) was cultured in 6 ml of culture medium (Chromosome Medium B, Biochrom, Berlin, Germany) with 5 mg/ml of phytohemagglutinin (Biochrom). Potassium tetraborate (K2B4O7, CAS Number 12045-78-2) was synthetized previously described by Marezio et al. (1963[15 ]) and dissolved in distilled water and it was added to the cultures just before incubation in concentrations of 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, 640 and 1280 µg/ml. Each individual blood culture without PTB was studied as a control group. The concentrations were selected according to the works of Turkez et al. (2007[25 ]). Mitomycin C (MMC; 10-7 M, Sigma-Aldrich®) was used as the positive control in CA and MN assays. Ascorbic acid (10 µM, Sigma-Aldrich®) and hydrogen peroxide (25 µM, Sigma-Aldrich®) were also used as the positive controls in TAC and TOS analysis, respectively.
10
Fanca and Fancc Knockout Mouse Model
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