Lentiviral particles were produced according to previously published protocols (50 (link)). In short, 1 day before transfection, 1.5 × 106 HEK293T cells [American Type Culture Collection (ATCC)] were plated on 100-mm dishes (Corning) in DMEM (PAA Laboratories) supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories) and 1× penicillin-streptomycin (PAA Laboratories). The transfection mix consisted of 300 μl of Opti-MEM (Thermo Fisher Scientific), 2.5 μg of transfer vector (pER4; Novartis), 2.5 μg of pCMV-ΔR8.2 (Addgene), 0.3 μg of pCMV-VSV-G (Addgene), and 20 μl of Fugene HD (Promega). Per construct, four dishes were prepared. One day later, the medium was changed. The next day, the supernatant was collected and filtered (0.45-μm pore size; Merck), and the viral particles were enriched using ultracentrifugation (Beckman) at ~140,000g. The pellet was resuspended in 150 μl of Opti-MEM, aliquoted, and stored at −80°C. For each experiment, the needed lentiviral particles were produced in batches to reduce intraexperimental deviations. Each batch was tested for comparable overexpression efficiencies, and the required transduction volumes were determined.
Opti mem
Manufactured by Beckman Coulter
Sourced in United States
Opti-MEM is a reduced serum media formulation developed by Gibco, a brand of Thermo Fisher Scientific. It is designed to support the growth and maintenance of various cell types, including mammalian cells, in cell culture applications. Opti-MEM is a chemically defined, serum-reduced medium that can be used for transfection, virus infection, and other cell culture experiments where reduced serum levels are desired.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Ultracentrifugation, by Beckman Coulter (1 mentions)
100 mm dishes, by Corning (1 mentions)
Pcmvδr8, by Addgene (1 mentions)
Pcmv vsv g, by Addgene (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Fugene hd, by Promega (1 mentions)
Sw28 swinging bucket rotor, by Beckman Coulter (1 mentions)
2 protocols using opti mem
1
Lentiviral Particle Production Protocol
2
Generating porcine induced pluripotent stem cells
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piPS-LCs were induced in accordance with a previously described protocol[16 (link)]. Briefly, human Oct4, Sox2, Klf4 and c-Myc were separately cloned into lentiviral vector FUGW (Addgene, 14883), and the constructs FUW-hOct4, FUW-hSox2, FUW-hKlf4 and FUW-hc-Myc were packaged into a virus in 293T cell lines by co-transfecting with auxiliary packaging vectors (psPAX2 and pMD2.G). After 2 days, the reconstructed lentivirus were collected and centrifuged in a SW28 swinging bucket rotor (Beckmann, USA) at 80,000 ×g for 2 h at 4°C, supernatant was then carefully removed and the pellets were suspended in Opti-MEM® reduced serum medium at 4°C overnight.
PFFs were seeded at 1 × 104 cells per well in a 12-well plate. On the next day, each concentrated virus was added to the medium with MOI (MOI = viral titer/cell number) ranging from 50 to 80 for each lentivirus and then incubated for 24 h. Three days after the initial viral transduction, the cells were digested and seeded onto MEF feeder layers. On day 4, PFF medium was replaced with porcine ESC medium for further culture. After 7–11 days, piPS colonies were harvested and plated onto new plates for further culture.
PFFs were seeded at 1 × 104 cells per well in a 12-well plate. On the next day, each concentrated virus was added to the medium with MOI (MOI = viral titer/cell number) ranging from 50 to 80 for each lentivirus and then incubated for 24 h. Three days after the initial viral transduction, the cells were digested and seeded onto MEF feeder layers. On day 4, PFF medium was replaced with porcine ESC medium for further culture. After 7–11 days, piPS colonies were harvested and plated onto new plates for further culture.
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