Lentivirus is produced by 293T cells. Specifically, 293T cells were seeded in a 6 cm dish (Thermo Fisher Scientific) before transfection. About 24 h later, the target vector, psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) were transfected together at a ratio of 4:3:1. The virus supernatants were collected 48 h and 72 h after the transient and filtered with a 0.45 μm membrane (Millipore) for usage.
6 cm dish
Manufactured by Thermo Fisher Scientific
The 6 cm dish is a laboratory equipment item used for culturing cells or performing other experimental procedures. It provides a contained environment with a flat surface area of approximately 28.27 square centimeters. The dish is typically made of polystyrene or other suitable materials.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
0.45 μm membrane, by Merck Group (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Vsv g, by Addgene (1 mentions)
Mirus lt1, by Mirus Bio (1 mentions)
0.45 μm syringe filter, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Polybrene, by Merck Group (1 mentions)
5 protocols using 6 cm dish
1
Lentivirus Production by 293T Cells
2
Lentiviral Transduction of Sarcoma Cells
3
Pulse-Chase RNA Labeling in HeLa Cells
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HeLa cells (female, RRID: CVCL_0030) were seeded at a density of 6 × 105 cells per 6-cm dish (Thermo Fisher Scientific) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic at 37°C. In the next day, the medium was replaced with fresh medium containing 150 μM BrU, and then the cells were precultured for 12 h to label pre-existing RNAs. After the preculture, the cells were washed twice with the medium, and then the medium was replaced with one containing 200 μM 4sU to label newly synthesized RNAs until 12 h. After incubating cells in the 4sU-containing medium, the cells were washed twice with PBS, and then total RNA was isolated using RNAiso Plus (TAKARA).
4
NADH and FAD Imaging Protocol
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For all experiments, cells were plated at 2.5×10 5 cells per 6 cm dish (Thermo Fisher Scientific Inc.) and images were obtained after 48 hours on a Nikon FN1 upright microscope equipped with an A1R scan head and Plan Apo LWD 25× waterimmersion objective lens NA 1.1. Images of different fluorophores were acquired sequentially using the two settings: NADH 405 nm excitation, 425-475 nm emission; and flavin adenine dinucleotide (FAD) 488 nm excitation, 500-550 nm emission. The 12-bit 2048×2048-pixel images were acquired, and final magnification was adjusted by zooming with the laser to attain appropriate pixel size.
5
BrU and 4sU RNA Labeling in HeLa Cells
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HeLa cells (female, RRID: CVCL_0030) were seeded at a density of 6×10 5 cells per 6-cm dish (Thermo Fisher Scientific) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic at 37ºC. After 12 h, the medium was replaced with fresh medium containing 150 μM BrU and then the cells were precultured for 12 h to label pre-existing RNAs. After the pre-culture, the cells were washed twice with the medium, and then the medium was replaced with one containing 200 μM 4sU to label nascent RNAs. After incubating cells in the 4sU-containing medium, the cells were washed twice with PBS and then total RNA was isolated using RNAiso Plus (TAKARA).
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