32D murine myeloid cells (#ACC 411) and Ba/F3 murine pro-B cells (#ACC 300) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). Cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM l -glutamine, 10 U mL–1 penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific) and 1 ng mL–1 murine Interleukin-3 (mIL-3; PeproTech). To generate retrovirus, Platinum-E retroviral packaging cells (Cell Biolabs) were transfected with plasmids using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific), as per the manufacturer’s protocols. 32D and Ba/F3 cells were transduced with viral particles by spinfection, and pools of cells stably expressing the transgenes of interest were selected after 48 h by sorting for GFP-positive cells using fluorescence-activated cell sorting (FACS).
Platinum e retroviral packaging cells
Manufactured by Cell Biolabs
Sourced in United States
The Platinum-E retroviral packaging cells are a high-performance cell line designed for the production of recombinant retroviruses. They are engineered to stably express the necessary retroviral packaging components, enabling the efficient generation of high-titer virus particles.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
N myc tagged pmxs puro retroviral vector, by Cell Biolabs (2 mentions)
Anti qki, by Fortis Life Sciences (2 mentions)
Anti myb antibody, by Abcam (2 mentions)
Anti myc hrp, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
P3000, by Thermo Fisher Scientific (1 mentions)
0.45 m low protein binding membrane, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hexadimethrine bromide polybrene, by Merck Group (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Fugene 6 transfection agent, by Promega (1 mentions)
Retro x concentrator, by Takara Bio (1 mentions)
Polybrene, by Merck Group (1 mentions)
10 protocols using platinum e retroviral packaging cells
1
Generation of Stable Transgenic Cell Lines
2
Production of Retroviral and Lentiviral Particles
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To make retroviral particles, a 10-cm culture dish of 80% confluent Platinum-E retroviral packaging cells (Cell Biolabs) was transfected in OptiMEM (Life Technologies) using 10 µg of the MSCV-based plasmid, 25 µL of P3000 (Life Technologies), and 25 µL of Lipofectamine 3000 (Life Technologies). After 6 h, culture medium was changed to DMEM (Life Technologies) with 10% FBS (Hyclone). After 48 h, viral supernatants were harvested and centrifuged at 1500 rpm for 5 min at 4°C to pellet cell debris. The supernatant was filtered through a 0.45-µm low-protein-binding membrane (Millipore) and used immediately or stored at −80°C until use.
To make lentiviral particles, pLenti-GFP, pLenti-Pax5, and pLenti-Arid3a (see “Plasmids”) were cotransfected with packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260) as described previously (Shalem et al. 2014 (link)). Briefly, a 10-cm culture dish of 80% confluent HEK293T cells was transfected in OptiMEM (Life Technologies) using 10 µg of the pLenti plasmid, 5 µg of pMD2.G, 7.5 µg of psPAX2, 25 µL of P3000 (Life Technologies), and 25 µL of Lipofectamine 3000 (Life Technologies). Lentiviruses were harvested as above.
To make lentiviral particles, pLenti-GFP, pLenti-Pax5, and pLenti-Arid3a (see “Plasmids”) were cotransfected with packaging plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260) as described previously (Shalem et al. 2014 (link)). Briefly, a 10-cm culture dish of 80% confluent HEK293T cells was transfected in OptiMEM (Life Technologies) using 10 µg of the pLenti plasmid, 5 µg of pMD2.G, 7.5 µg of psPAX2, 25 µL of P3000 (Life Technologies), and 25 µL of Lipofectamine 3000 (Life Technologies). Lentiviruses were harvested as above.
3
Retroviral Packaging Cell Line Protocol
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Platinum-E retroviral packaging cells (Cell biolabs, #RV-101) were maintained in DMEM, 10% FCS with penicillin-streptomycin, supplemented with puromycin (1 mg ml-1) and blasticidin (10 mg ml-1). On the day before transfection, 3 million cells were seeded in a 100 mm culture dish in 10 ml of media without antibiotics. Cells were transfected at 70% confluency using Fugene HD Transfection Reagent (Promega). For each 100 mm culture dish, 950 ml OPTI-MEM (GIBCO) was mixed with 11 mg pCl-Eco, 22 mg library plasmid and 99 ml Fugene HD. The transfection mixture was incubated for 10 min at room temperature prior to addition. At 18 h post-transfection, the media was replaced with 10 ml fresh media, and viral supernatant was harvested at 48 and 72 h post-transfection. Cells were removed by filtering through a 0.45 mm syringe filter.
4
Immortalization of Mouse Embryonic Fibroblasts
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Timed matings were performed between heterozygous mice. Pregnant females were culled at E12.5 to harvest embryos. Embryos were incubated in pre-warmed trypsin solution (2.5 μg.mL−1 trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH2PO4, 25 mM glucose, 25 mM EDTA, pH 7.6) for 10 min and disaggregated by gentle pipetting. Primary mouse embryonic fibroblast (MEF) cultures were established following standard methods and immortalized using the SV40 large T antigen as described previously. Briefly, Platinum-E retroviral packaging cells (Cell Biolabs) were transfected with pBABE-SV40-Puro and the culture media containing the virus was harvested 48 h later and passed through a 0.22 μm filter. The filtered retrovirus was mixed 1:1 with complete MEF media supplemented with 1 μg.mL−1 hexadimethrine bromide (Polybrene, Millipore). The infective medium was subsequently added to primary MEF cultures and transformed clones were selected for 14 days using 1 μg.mL−1 puromycin.
5
Retroviral Expression of MYB-QKI Fusion Constructs
6
Generating Stable Cell Lines for Gene Silencing and Overexpression Studies
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For the gene-silencing experiments, platinum-E retroviral packaging cells (Cell Biolabs; San Diego, CA, USA) were transfected with mouse PRSS8 shRNA (Silencer Select shRNA, ID: S94451, Thermo Fisher Scientific) or control shRNA (ID: 4,390,844) using jetPRIME (Polyplus; New York, USA) as described by the manufacturers’ instructions. MIN6 cells were infected with the retroviral supernatant after 48 h and selected with 1 mg/mL puromycin for 1–2 weeks. Colonies were picked to generate monoclonal cell lines.
For the gene-overexpressing experiments, MIN6 cells were transfected with pcDNA3.1-PRSS8 (GenScript, ID: OHu16476), pcDNA3.1-mutant PRSS8 (alanine substitution at the active site), or pcDNA3.1-empty vector using jetPRIME.
For the transient transfection experiments, cells were harvested 48 h after transfection, and PRSS8 expression was determined by western blotting. Cells were selected with 25 mg/mL hygromycin using the same technique to generate stable cell lines.
For the gene-overexpressing experiments, MIN6 cells were transfected with pcDNA3.1-PRSS8 (GenScript, ID: OHu16476), pcDNA3.1-mutant PRSS8 (alanine substitution at the active site), or pcDNA3.1-empty vector using jetPRIME.
For the transient transfection experiments, cells were harvested 48 h after transfection, and PRSS8 expression was determined by western blotting. Cells were selected with 25 mg/mL hygromycin using the same technique to generate stable cell lines.
7
Retroviral Expression of MYB-QKI Fusion Constructs
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MYB-QKI5 and MYB-QKI6 constructs were synthesized as Gateway compatible entry clones. MYBtr constructs were generated via PCR mutagenesis using MYB-QKI fusions as templates. Full-length MYB and QKI constructs were purchased as gateway entry clones from PlasmID/DF/HCC DNA Resource Core. MYB-QKI5 and MYB-QKI6, MYBtr, full-length MYB and QKI constructs were sub-cloned into a Gateway-compatible N-MYC-tagged pMXs-Puro Retroviral Vector (Cell Biolabs). Platinum-E retroviral packaging cells (Cell BioLabs) were used to generate retrovirus as per manufacturer protocols. NIH3T3 cells were infected with retrovirus containing media for 6 hours and puromycin selection commenced 48 hours post infection. Stable expression of MYC-tagged proteins was confirmed via western blot analysis (anti-MYC HRP 1:5000 (Invitrogen), anti-MYB antibody 1:5000 (Abcam) and anti-QKI 1:1000 (Bethyl Lab).
8
Retroviral Transduction of JAK2 Variants
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Murine JAK2WT (WT) and murine JAK2V617F cDNA15 (link) were cloned into the retroviral vector pMCs-IRES-GFP (Cell Biolabs, San Diego, CA, USA). Transient transfection of Platinum-E retroviral packaging cells (Cell Biolabs) was performed by using the FuGENE6 transfection agent (Promega, Madison, WI, USA) according to the manufacturer's protocol. Retroviral supernatants were harvested after 48 h and used to transduce the murine interleukin (IL)-3 -dependent pro-B cell line Ba/F3 or bone marrow cells.
9
Engineering Gallbladder Organoids with ERBB2
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Gallbladder organoids derived from KraslslG12D mice were transiently cotransfected with pt3-PGK-Blasticidin-P2A-EGFP and either U6-sgCr8-EFS-Cas9-P2A-Cre (KCR8 organoids), U6-sgp53-EFS-Cas9-P2A-Cre (KP organoids) or U6-sgp53-U6-sgPten-EFS-Cas9-P2A-Cre (KPP organoids) using Lipofectamine2000 (ThermoFisher Scientific, Waltham, MA, USA) and selected with blasticidin (20 µg/mL). Prior to transduction with different ERBB2 expressing retroviruses we transfected gallbladder organoids from C57BL/6J mice with px459_sgp53 and selected with puromycin (50 µg/mL).
To mark organoids with a green fluorescent marker (EGFP), we cotransfected pt3-PGK-Blasticidin-P2A-EGFP with the sleeping beauty-13 plasmid (kindly provided by David A. Largaespada, University of Minnesota, Minneapolis, MN, USA) using Lipofectamine2000 and selected with blasticidin (20 µg/mL). MSCV-based retroviruses (pMSCV-ERBB2-IRES-EGFP) were produced in Platinum-E retroviral packaging cells (Cell Biolabs, San Diego, CA, USA), concentrated using Retro-X concentrator (Clontech, Mountain View, CA, USA), and supplemented with polybrene (4 µg/mL) prior to transduction of organoids.
To mark organoids with a green fluorescent marker (EGFP), we cotransfected pt3-PGK-Blasticidin-P2A-EGFP with the sleeping beauty-13 plasmid (kindly provided by David A. Largaespada, University of Minnesota, Minneapolis, MN, USA) using Lipofectamine2000 and selected with blasticidin (20 µg/mL). MSCV-based retroviruses (pMSCV-ERBB2-IRES-EGFP) were produced in Platinum-E retroviral packaging cells (Cell Biolabs, San Diego, CA, USA), concentrated using Retro-X concentrator (Clontech, Mountain View, CA, USA), and supplemented with polybrene (4 µg/mL) prior to transduction of organoids.
10
Retroviral Transduction of Naive CD8+ T Cells
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Retroviral constructs were created by insertion of TOX, M1T, or M (40, 42, 44 )I sequences into p-MIG-eGFP retroviral vectors. Platinum-E Retroviral Packaging Cells (Cell Biolabs, Inc., San Diego, CA) were transfected in a 100 mm dish using Fugene HD Transfection Reagent (Promega, Madison, WI) with 10μgs of plasmid DNA. Viral supernatant was collected after 48 hours. On day 0, naïve CD8 + T cells were obtained via negative selection of OTI splenocytes and cultured in complete RPMI with IL-7, IL-15, and OVA peptide as above. 24 hours later, cells were washed, and resuspended in viral supernatant supplemented with IL-7, IL-15, and 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO). Cells were spin infected at 34°C for 75 minutes at 2000xg. Cells were returned to the incubator for 4 hours, washed, and resuspended in complete media with cytokines and OVA as above. Cells were treated with OVA again on day 2, media was changed on day 3, and cells collected for analysis on day 5.
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