All cell lines were grown at 37 °C under standard cell culture conditions (humidified atmosphere, 5% CO2). ALT-positive GM847, SW26, and U-2 OS cells, as well as derived cell lines, and ALT-negative HeLa, HeLa LT (long telomeres), SW39 and hTERT-RPE1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (GIBCO) and penicillin-streptomycin antibiotics. For U-2 OS CycE/RAD52 WT and KO cells G418 400 μg/ml (Gibco, 10131-027), puromycin 1 μg/ml (Sigma, P8833) and tetracycline 2 μg/ml (Sigma, T7660) were added to the medium. U-2 OS 53BP1-GFP/RPA70-mScarlet cells were maintained in presence of puromycin 0.5 μg/ml (Sigma, P8833) and 5 μg/ml blasticidin (InvivoGen, ant-bl-05). All cells used in this study were grown under sterile conditions and routinely (monthly) tested for mycoplasma contamination and scored negative. The following compounds were used in this manuscript at the indicated final concentrations unless stated otherwise: ATRi Az-20 (5 μM for RPE-1 cells, else 1 μM, Tocris), ATRi VE-821 (5 μM, Selleckchem), APH (0,2 μM, Sigma-Aldrich), HU (2 mM, Sigma-Aldrich), Nocodazole (50 ng/ml, Sigma-Aldrich), CDKi RO-3306 (9 μM, Sigma-Aldrich), Colcemid (0,1 μg/ml, Roche), ATMi KU-55933 (10 μM, Selleckchem).
Colcemid
Manufactured by Roche
Sourced in Switzerland, United States, Germany
Colcemid is a chemical compound used in laboratory research and cell biology applications. It acts as a mitotic inhibitor, preventing the formation of the mitotic spindle during cell division. Colcemid is commonly used to arrest cells in metaphase, facilitating the analysis of chromosomes and cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Nocodazole, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Giemsa, by Merck Group (3 mentions)
Thymidine solution, by Merck Group (2 mentions)
Metafer automated scanning and imaging platform, by MetaSystems (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Blocking reagent, by Roche (2 mentions)
Orca er camera, by Hamamatsu Photonics (2 mentions)
Thymidine, by Merck Group (2 mentions)
Ro 3306, by Selleck Chemicals (2 mentions)
Metafer imaging platform, by MetaSystems (2 mentions)
Axio imager d2 microscope, by Zeiss (2 mentions)
Telomere repeat specific peptide nucleic acid probes, by Thermo Fisher Scientific (2 mentions)
Plan apo 1.4na, by Zeiss (2 mentions)
Lipopolysaccharide solution, by Merck Group (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Merck Group (2 mentions)
Phytohemagglutinin m, by Thermo Fisher Scientific (2 mentions)
67 protocols using colcemid
1
Maintaining and Characterizing ALT and Telomere-related Cell Lines
2
Chromosome-specific CO-FISH Assay
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The CO-FISH assay was adapted from previous studies [35 (link), 36 (link)]. Briefly, exponentially growing cells were cultured overnight in the presence of 10 μM BrdU:BrdC (3:1) (Sigma-Aldrich) at 37 °C to allow for one round of replication. Colcemid (Roche) was then added at a concentration of 0.1 μg/ml for 4 h to arrest cells at prometaphase. After fixation of cellular preparations on slides and Hoechst 33258 (Sigma-Aldrich) staining, the newly synthesized strands were degraded following UV light exposure and treatment with 10 U/μl ExoIII (Promega). Hybridization was performed using fluorescent centromeric PNA probes against CENP-B box motif sequences. The PNA probe labeled with Cy3 (ATTCGTTGGAAACGGGA; PNABio Inc) hybridizes with the leading strand and the reverse PNA probe labeled with Alexa-488 (TCCCGTTTCCAACGAAT; Eurogentec) hybridizes with the lagging strand. DNA was counterstained with DAPI (Sigma-Aldrich). Metaphases were captured on a confocal laser microscope (FV1000 Olympus) and images were analyzed using Image J software (NIH). Quantitation to measure for SCE between α satellite sequences (C-SCE) was done by counting the number of CO-FISH signals showing C-SCE over the total number of CO-FISH signals observed for each metaphase.
3
Chromosome Analysis of Induced Pluripotent Stem Cells
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iPS cells were plated on Matrigel-coated T25 flasks and cultivated for 2–3 days in mTeSR1 medium. Chromosome analysis was carried out according to established protocols. In brief, cells were synchronized using thymidine solution (Sigma-Aldrich) and subsequently treated with colcemid (Roche) for 10 min at 37°C. After detachment with trypsin—EDTA, cells were centrifuged, and the cell pellet was re-suspended and maintained in hypotonic solution (75 mM KCl) for 12 min at 37°C. Cells were then fixed in methanol and acetic acid. Metaphase spreads were prepared on cover slips, dried overnight and Giemsa stained (Sigma-Aldrich) after trypsin pre-treatment.
4
Karyotyping of Cultured Cell Lines
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For karyotyping, cells were seeded on gelatine and passaged three times before harvesting. After reaching 50 to 60% confluence, cells were treated with 0.1 μg/ml colcemid (Roche, #10295892001) for 2 hours in the incubator. Cells were trypsinized and transferred to a 15 ml Falcon tube and collected by centrifugation at 200 x g for 4 min. Hypotonic treatment in 0.4% KCl was performed for 10 min at 37°C in a water bath. At the end of hypotonic treatment, 100 μl of fresh-made fixative (3:1 methanol:acetic acid) was added and mixed by inversion. Cells were collected by centrifugation at 200 x g for 4 min and 5 ml of fixative was added to the pellet for 20 min at room temperature. After centrifugation the pellet was resuspended in 500μl of fresh-made fixative. Two drops were spotted on a pre-chilled slide for a quality check under the microscope. At least 15 metaphases each were counted for the seven independent clones. Four clones had the correct number of 40 chromosomes (S1 Table ).
5
Mitotic Chromosome Spread Analysis
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Cells were treated with DMSO or 0.5 μM dTAG-13 for 18 h and subsequently arrested at mitosis with 0.1 mg/ml colcemid (Roche) for 6 h (total 24 h). Metaphase chromosome spreads were prepared as previously described (Zong et al, 2019 (link); Zong et al., 2015 (link)). First, cells were induced to swell in a prewarmed hypotonic solution containing 0.75 M KCl (Sigma) for 20 min at 37°C. Next, suspensions of single cells were fixed with a solution mixture containing methanol and glacial acetic acid at a 3:1 ratio. Thereafter, cells were further washed extensively with the same fixative solution and dropped onto slides in a humidified chamber (Thermotron Industries). To visualize metaphase chromosomes by fluorescence in situ hybridization (FISH), samples immobilized on slides were sequentially treated with pepsin (5–10 μg/ml in 0.01 N HCl, 5 min at 37°C), washed, dehydrated with ethanol, briefly heat denatured (80°C, 1 min 15 s) in a slide moat (Boekel Scientific) and incubated with a commercially available Cy3-labeled (CCCTAA)3 peptide nucleic acid probe (PNA Bio) recognizing mammalian telomere sequences. After extensive washes, DNA was counterstained with DAPI. Images were acquired at 63x magnification using the Metafer automated scanning and imaging platform (MetaSystems). Fifty individual metaphases were scored for the presence of chromosomal aberrations.
6
Analysis of Murine Bone Marrow Cells
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Total bone marrow cells were extracted from femurs and tibias of mice using PBS supplemented with 1% FBS and 1 mM EDTA. After lysing red blood cells, nucleated cells were cultured in RPMI-1640-rich medium supplemented with 10% FBS and 50 μM β-mercaptoethanol, recombinant murine Flt3-ligand (20 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), SCF (20 ng/ml), TPO (50 ng/ml), and recombinant human TSLP (10 ng/ml). After 24 h, bone marrow cells were exposed to 2 Gy IR (X-RAD 320). One hour before harvesting cells, 150 ng/ml of colcemid (Roche, Basel, Switzerland) was added to the culture. Cells were then harvested, treated in 75 mM potassium chloride (KCl) at 37 °C for 20 min, and fixed with ice-cold methanol/acetic acid (3:1) fixative. Fixed cells were then dropped on slides to generate metaphase spreads. Slides were allowed to dry overnight and then mounted with Vectorshield containing DAPI (Vector Laboratories, Burlingame, CA, USA) for analysis. Images were taken with AxioImager Z1 (Zeiss, Oberkochen, Germany) equipped with Metamorph software (Molecular Devices, Sunnyvale, CA, USA).
7
High-Resolution Karyotyping of Dermal Fibroblasts and hiPSCs
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For numerical and structural chromosome analyses of dermal fibroblasts and hiPSCs, high-resolution karyotyping was performed. Fibroblasts were grown in T25 culture flasks in fibroblast growth medium. Human iPSCs were cultivated in T25 flasks coated with hESC-qualified Matrigel in mTeSR™1 maintenance medium. Cells were synchronised using thymidine solution (Sigma-Aldrich, Munich, Germany) and subsequently treated with colcemid (Roche, Mannheim, Germany) for 10 min at 37 °C. After detachment with trypsin–EDTA, cells were centrifuged, and the cell pellet was re-suspended and maintained in hypotonic solution (75 mM KCl) for 12 min at 37 °C. Cells were then fixed in methanol and acetic acid. Metaphase spreads were prepared on cover slips, dried overnight and Giemsa stained (Sigma-Aldrich, Munich, Germany) after trypsin pre-treatment.
8
Assessing PARP Inhibitor Effects
9
Cytogenetic Analysis of Danusertib Effects
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Conventional cytogenetics techniques were performed as described in [7 (link)] in order to determine the effect of 48 h of treatment with 500 nM Danusertib on the mitotic index and cell ploidy. Briefly, cells were treated with 0,2 μg/ml of Colcemid (Roche) and then harvested and incubated with a hypotonic solution of 0,56% w/v of KCl for 15 minutes at room temperature. Subsequently, cells were fixed with a fixative solution composed of 3:1 methanol:acetic acid. Chromosomes were QFQ banded using quinacrine mustard (Roche) and slides were mounted in McIlvaine buffer. Slides were analysed using Nikon Eclipse 80i fluorescence microscope (Nikon) equipped with a COHU High Performance CCD camera. The mitotic index was evaluated counting the percentage of mitosis scoring at least 1000 nuclei, while ploidy was investigated by evaluating the number of chromosomes/metaphase on 30/50 metaphases. Chromosomes spreads were analysed using the Genikon software. Data were obtained as mean values derived from two independent experiments.
10
Spectral Karyotyping of CAL-33 Cell Line
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Metaphase chromosome spreads were prepared from untreated CAL-33 parental cell line and generated CAL-33 sublines. Colcemid (Roche) was added at a final concentration of 0.1 μg/ml to the culture medium of exponentially growing cells at a density of 6 × 106 cells per 75 cm2. After 3 h of incubation time, cells were washed with PBS, trypsinized, suspended in fresh culture medium followed by hypotonic KCl treatment (75 mM) at 37 °C for 25 minutes. Following centrifugation, cells were resuspended in 2–3 ml of fixation solution and approximately 40–50 μl of cell suspension was dropped on several microscope slides. After one week of ageing at room temperature, spectral karyotyping was performed as described by Hieber et al. [14 (link)]. The karyotype of each cell line was determined based on a minimum of 15 metaphases. Chromosomal aberrations were detectable by color junctions within affected chromosomes. Spectral imaging and image analysis were performed with a SpectraCube system and SkyView imaging software (both from Applied Spectral Imaging).
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