The NF-κB/Jurkat/GFPTM Transcriptional Reporter cell line was obtained from System Biosciences, and Raji B cells were obtained from the American Type Culture Collection. NF-κB reporter Jurkat cells and Raji cells were cultured in RPMI-1640 media supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. HEK293 LTV cells (Cell Biolabs Inc.), used to generate lentivirus, were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Human peripheral blood mononuclear cells (PBMCs) were a generous gift from the laboratory of Susan Kaech (Yale University). PBMCs were cultured in RPMI-1640 media supplemented with 10% FBS, 2 mM GlutaMAX (ThermoFisher Scientific), 50 μM beta-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin. NF-κB reporter Jurkat, Raji, HEK293 LTV, and PBMCs were cultured at 37°C with 5% CO2 in a humidified environment. Expi293F cells used for protein expression (ThermoFisher Scientific) were cultured in Expi293 media (ThermoFisher Scientific) at 37°C in a humidified environment containing 8% CO2 while shaking.
Hek293 ltv cells
Manufactured by Cell Biolabs
Sourced in United States
HEK293 LTV cells are a genetically modified cell line derived from human embryonic kidney (HEK) 293 cells. These cells are designed to express the large T antigen (LT) from the SV40 virus, which can enhance viral transduction and gene expression. The core function of HEK293 LTV cells is to serve as a reliable host for the production and amplification of lentiviral vectors.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Nf κb jurkat gfptm transcriptional reporter cell line, by System Biosciences (2 mentions)
Expi293f cells, by Thermo Fisher Scientific (2 mentions)
Expi293 media, by Thermo Fisher Scientific (2 mentions)
Raji b cells, by American Type Culture Collection (2 mentions)
Plp vsvg, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mc38 cells, by Kerafast (1 mentions)
Penicillin streptomycin, by Gemini Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Daudi, by American Type Culture Collection (1 mentions)
Amphotericin, by Lonza (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Amphotericin, by Corning (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
L glutamine, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
10 protocols using hek293 ltv cells
1
Cell Culture Conditions for NF-κB Studies
2
Lentiviral Vector Production Protocol
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In order to produce the relative lentiviruses, HEK-293LTV cells (Cell Biolabs, San Diego, CA, USA) were initially plated into a p15 dish and then transfected when ~ 90% confluent with 9 µg of pMD2G (envelop vector), 24 µg of pPAX2 (packaging vector) and 30 µg of the relative trans-vector in the presence of 1 mg/ml of polyethylenamine (PEI). 12 h after transfection, media were refreshed. 24 h later, media were collected and stored at 4 °C while the cells received new media. This procedure was repeated 24 h later and then media for the same lentivirus were pooled. After spinning at 2000 × g for 10 min at 4 °C to precipitate cells debris, 5X PEG-IT Virus Precipitation Solution (SBI System Biosciences, Mountain View, CA, USA) was added to the media. After vortexing, media were left at 4 °C overnight (at least 12 h). The day after, media were spun at 1500 × g for 30 min at 4 °C. Pellets (lentivirus particles) were suspended with PBS + 25 mM Hepes in 1/1000 of the initial volume of the pooled media. Aliquots were created and stored at −80 °C. Lentivirus titers were biologically established by qPCR.
3
Lentiviral Vector Production for EGFP and hOCT4
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Lentiviral vector plasmids coding enhanced green fluorescent protein (EGFP)
and human OCT4 (hOCT4) were prepared. EGFP and
hOCT4 inserts are obtained by PCR amplification from pLL3.7 (Invitrogen) and
FU-tet-o-hOCT4 (Addgene, Cambridge, MA, USA), respectively. Each insert was passed to the
LIN28 coding region of the pSIN-EF2-LIN28-PUR plasmid (Addgene). Preparation of lentivirus
particles followed the previous report [26 (link)] with modification. Prepared
self-inactivating lentiviral vector plasmids harboring transgenes, packaging plasmids (pLP1 and pLP2;
Invitrogen), and envelop plasmids (pLP/VSVG; Invitrogen) were used for lentivirus particle production, and
HEK293 LTV cells (Cell Biolabs, USA) were used for packaging lentivirus particles. The detailed procedure is
described in theSupplementary Materials and
Methods (online only).
and human OCT4 (hOCT4) were prepared. EGFP and
hOCT4 inserts are obtained by PCR amplification from pLL3.7 (Invitrogen) and
FU-tet-o-hOCT4 (Addgene, Cambridge, MA, USA), respectively. Each insert was passed to the
LIN28 coding region of the pSIN-EF2-LIN28-PUR plasmid (Addgene). Preparation of lentivirus
particles followed the previous report [26 (link)] with modification. Prepared
self-inactivating lentiviral vector plasmids harboring transgenes, packaging plasmids (pLP1 and pLP2;
Invitrogen), and envelop plasmids (pLP/VSVG; Invitrogen) were used for lentivirus particle production, and
HEK293 LTV cells (Cell Biolabs, USA) were used for packaging lentivirus particles. The detailed procedure is
described in the
Methods
4
Luciferase-Labeled Cell Lines for In Vivo Research
5
Cell Line Culturing and Lentivirus Production
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K562, Daudi, Jurkat, and Raji cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Raji/Luc-GFP cells were a gift from Dr. Yvonne Y. Chen at the University of California, Los Angeles [28 (link)]. All cell lines were cultured in RPMI-1640 supplemented with 10% dialyzed fetal calf serum (FCS) (dFCS, Hyclone, Logan, UT, USA), 2% glutamine and 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, USA). HEK-293 LTV cells (Cell Biolabs Inc., San Diego, CA, USA) cultured in DMEM (Corning, Manassas, VA, USA) supplemented with 10% dFCS, 2% glutamine and 1% pen/strep were used for lentivirus production.
6
Cell Line Culture and Authentication
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SW480, LS174T, and CT26 cell lines were obtained from ATCC. HEK-293LTV cells were obtained from Cell Biolabs. LS174T, HEK-293LTV, and CT26 cells were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% v/v fetal bovine serum (Corning), L-glutamine (2 mM; Gibco), penicillin-streptomycin (100 U/ml; Gibco), Amphotericin (1 μg/ml; Lonza), and sodium pyruvate (1 mM; Gibco). SW480 cells were grown in McCoy’s 5A modified media with L-glutamine (Corning) supplemented with 10% v/v fetal bovine serum, penicillin-streptomycin (100 U/ml), Amphotericin (1 μg/ml), and sodium pyruvate (1 mM). All cells were grown at 37°C under 5% CO2 and passaged when the monolayer reached 80% confluency. All cell lines were authenticated by SPR profiling at MSKCC. All cells were regularly checked for mycoplasma contamination and have been negative.
7
Lentiviral Particle Production Protocol
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Recombinant lentiviral particles were produced as described previously (Lois et al. 2002) . In brief, HEK293LTV cells (Cell Biolabs, San Diego, USA) were transfected with equimolar amounts of pFSGW, pdelta8.9 and pVSVg using polyethyleneimine (Sigma-Aldrich, St. Louis, USA). Virus particles were harvested two days after transfection by collecting the cell culture supernatant and concentrated by ultracentrifugation (90 min at 75,000 g in a SW32Ti rotor, Beckman Coulter, Brea, USA). Virus-containing solution was aliquoted, shock frozen in liquid nitrogen and stored at -80 °C for further use.
8
Lentiviral Vector Production Protocol
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Lentiviral vectors were produced as previously described [21 ]. Briefly, HEK 293 LTV cells (Cell Biolabs, San Diego, CA, USA) were used as the packaging cell line, and five plasmids were used for the production of lentiviral vectors: FUW-tetO-pMYOD1 and FUW-M2rtTA (the transfer plasmid); pLP1 and pLP2 (the packaging plasmids; Invitrogen, Waltham, MA, USA); and pLP/VSVG (the envelope plasmid; Invitrogen). These plasmids were transfected into HEK 293 LTV cells using the calcium phosphate precipitation method. Subsequently, the LTV culture supernatants were filtered and concentrated. The derived virus pellets were stored at − 76 °C until use.
9
Culture of HEK293 and Neural Stem Cells
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HEK293 LTV cells (Cell Biolabs) were cultured in DMEM medium (Gibco), supplemented with 10% FBS at 37 °C, 5% CO2. Human neural stem cells (NSCs) (Gibco) were cultured in NSC medium (KnockOut DMEM-F12 (Gibco), 2 mM L-glutamine (Gibco), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Peprotech), 2% StemPro Neural supplement (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin), as previously described [87 (link)].
10
Cell Culture Protocols for Immunological Studies
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The NF-κB/Jurkat/GFP TM Transcriptional Reporter cell line was obtained from System Biosciences, and was cultured in RPMI-1640 media supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C with 5% CO 2 in a humidified environment.
HEK293LTV cells (Cell Biolabs Inc.) used to generate lentivirus were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C in 5% CO2 in a humidified environment. Human peripheral blood mononuclear cells (PBMCs) were a generous gift from Susan Kaech. PBMCs were cultured in RPMI 1640 media supplemented with 10% FBS, 2 mM GlutaMAX (ThermoFisher Scientific), 50 μM beta-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C with 5% CO 2 in a humidified environment. Raji B cells were obtained from American Type Culture Collection and were cultured in RPMI-1640 media supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C with 5% CO 2 in a humidified environment. Expi293F cells used for protein expression (ThermoFisher Scientific) were cultured in Expi293 media (ThermoFisher Scientific) at 37˚C in a humidified environment containing 8% CO 2 while shaking.
HEK293LTV cells (Cell Biolabs Inc.) used to generate lentivirus were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C in 5% CO2 in a humidified environment. Human peripheral blood mononuclear cells (PBMCs) were a generous gift from Susan Kaech. PBMCs were cultured in RPMI 1640 media supplemented with 10% FBS, 2 mM GlutaMAX (ThermoFisher Scientific), 50 μM beta-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C with 5% CO 2 in a humidified environment. Raji B cells were obtained from American Type Culture Collection and were cultured in RPMI-1640 media supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37˚C with 5% CO 2 in a humidified environment. Expi293F cells used for protein expression (ThermoFisher Scientific) were cultured in Expi293 media (ThermoFisher Scientific) at 37˚C in a humidified environment containing 8% CO 2 while shaking.
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