Pseudoviral particles were produced as described previously [68 (link)]. In brief, 293-TN cells (System Biosciences, SBI) were co-transfected with the lentiviral plasmid and lentiviral helper plasmids (psPAX2 and pMD2.G, gifts from Didier Trono) using PureFection (SBI), following manufactures protocol. Retroviral particles were produced using 293-GPG cells, which contain the viral packaging plasmid inside. 293-GPG cells were also transfected with plasmid of interest using PureFection. Lentivirus or retrovirus containing supernatants were collected at 48 hours and 72 hours after transfection, and filtered through a 0.45 μm filter. Cells were seeded into 6-well plates so that they reached 50–60% confluency on the day of infection and transduced at most 3 consecutive days with the viral stock in the presence or absence (for BT-549 cell line) of 8 μg/mL polybrene (Sigma-Aldrich). The viral solution was replaced with fresh culture medium, and cells were cultured for 24 hours before selection with 1 μg/mL of puromycin (InvivoGen) or 6ug/mL of blasticidine S (InvivoGen) for 10 days. Cells were also sorted using fluorescence assisted cell sorting (FACS) to ensure expression of all the fluorescent reporters and maintained in a media with antibiotics to ensure continued plasmid expression.
Blasticidine s
Manufactured by InvivoGen
Sourced in United States, Sweden
Blasticidine S is a selective antibiotic used for the detection and selection of transfected cells. It acts by inhibiting protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by InvivoGen (2 mentions)
Plasmocin, by InvivoGen (2 mentions)
Polybrene, by Merck Group (1 mentions)
293tn cells, by System Biosciences (1 mentions)
Purefection, by System Biosciences (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Geneticin, by InvivoGen (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Phleomycin, by InvivoGen (1 mentions)
Hygromycin b, by InvivoGen (1 mentions)
4 protocols using blasticidine s
1
Lentiviral and Retroviral Particle Production
2
Cell Culture Protocol for Diverse Cell Lines
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Mock and Flag-WRAP53β U2OS cells were maintained in McCoy's 5A medium (HyClone, Thermo Scientific), selected with 10μg/ml Blasticidine S (InvivoGen), U2OS cells were maintained in McCoy's 5A medium (HyClone, Thermo Scientific), U2OS-FokI cells were maintained in DMEM with GlutaMAX (Gibco, Life Technologies) and H1299 cells (human non-small cell lung carcinoma cell line) were maintained in Dulbecco's modified medium (HyClone, Thermo Scientific) supplemented with 10% fetal bovine serum (HyClone) and 2,5 µg/mL Plasmocin (InvivoGen) at 37°C in 5% CO2 humidified incubators. The stable Flag-WRAP53β cells overexpress the open-reading frame of the protein tagged with 1xFlag.
3
Antibiotic Resistance Screening of Yeast
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A variety of antibiotics was added to the YPD agar to assess the antibiotic resistance for strain SMB. A single colony was inoculated into 10 mL YPD broth (incubated at 30 °C with 250 rpm) and 100 µL of the overnight yeast culture was spread on several YPD plates supplemented with different types of antibiotics at respective concentrations for 3 days. The antibiotics and the recommended concentrations used were as follows: Blasticidine-S (InvivoGen, San Diego, CA, USA) (50 μg/mL), Zeocin (500 μg/mL), Hygromycin B (50 μg/mL), Puromycin (InvivoGen, San Diego, CA, USA) (25 μg/mL), Geneticin (InvivoGen, San Diego, CA, USA) (400 μg/mL), and Phleomycin (InvivoGen, San Diego, CA, USA) (25 μg/mL).
4
U2OS cell lines for HR and NHEJ assays
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Mock and Flag-WRAP53β U2OS cells were maintained in McCoy's 5A medium (HyClone, Thermo Scientific, Stockholm, Sweden), selected with 10 μg/ml blasticidine S (InvivoGen), and DR-GFP and EJ5-GFP U2OS cells were maintained in high glucose DMEM (HyClone), supplemented with 10% fetal bovine serum (Hyclone) and 2.5 μg/ml plasmocin (InvivoGen) at 37 °C in 5% CO2 humidified chamber. Flag-WRAP53β cells overexpress the open-reading frame of the protein tagged with 1xFlag.
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