Lentiviral particles were generated by using 3rd generation lentiviral system. Four plasmids (packaging: pMD2G, envelop: pMDLg/pRRE and pRSV-Rev and target vector: pHIV-EGFP) were procured from Addgene, (Plasmid repository, USA). Lentiviral packaging cell line (Lenti-X 293T cells) procured from Clontech (USA). Lenti-X 293T cells were transfected with Lipofectamine 3000 (Invitrogen, USA) and lentivirus particles were produced with titer of 2.8 × 105 TU per ml. Next, 5 × 105 HPKs obtained from TCP and 20 kPa gel were seeded in 24-well plate in 500 μl of keratinocyte growth medium. Next day, lentivirus particles (MOI-0.5) were added in the presence of polybrene and incubated for 24 h. One well was kept as a control without virus addition. Post 24 h, 1 ml keratinocytes growth medium was added in each well and incubated for next 48 h. Percentage transduction efficiency was measured by GFP fluorescent intensity in BD FACSVerse flow cytometer.
Lenti x 293t cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lenti-X 293T cells are a human cell line derived from 293T cells, which are commonly used for the production of lentiviral particles. These cells are suitable for the transfection and expression of lentiviral packaging plasmids, allowing for the efficient generation of recombinant lentiviral particles.
Automatically generated - may contain errors
Lab products found in correlation
Lenti x 293t cells, by Takara Bio (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Pspax2, by Addgene (2 mentions)
Pmd2 g, by Addgene (2 mentions)
3t3 l1 cells, by American Type Culture Collection (1 mentions)
Oleic acid palmitic acid, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Dulbecco s modified, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mdck cells, by RIKEN BioResource Center (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hcc70, by American Type Culture Collection (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
9 protocols using lenti x 293t cells
1
Lentiviral Transduction of Human Primary Keratinocytes
2
Establishing NAFLD Model in HepG2 Cells
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HepG2 cells, 3T3L1 cells and Lenti-X 293T cells were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10% fetal bovine serum (HyClone) and penicillin/streptomycin (100 U/ml, Invitrogen). To establish an NAFLD model in HepG2 cells, 0.1 mM oleic acid (Sigma-Aldrich), or 0.5 mM FFAs (oleic acid/palmitic acid (Sigma-Aldrich), 2:1) for 24 h74 (link). In some experiments, HepG2 cells were cultured with 25 µM chloroquine (Sigma-Aldrich) for 24 h or 5 nM Bafiromycin A1 (Sigma-Aldrich) for 2 h75 (link). HepG2 cells and 3T3L1 cells were purchased from ATCC, and Lenti-X 293T cells were from Clontech. No commonly misidentified cell line was used in this study. All the cell lines were routinely tested negative for mycoplasma contamination.
3
Cell Line Maintenance Protocol
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MDCK cells were purchased from the RIKEN BioResource Center (no. RCB0995). Lenti-X 293T cells were obtained from Invitrogen. MDCK and Lenti-X 293T cells were maintained in Dulbecco’s modified Eagle medium (FUJIFILM Wako Pure Chemical Corp.) containing 10% fetal bovine serum (Sigma-Aldrich) and 1% v/v penicillin−streptomycin (Nacalai Tesque). Cell line identity were validated.
4
Lentiviral Transduction of Human Primary Keratinocytes
5
Lentiviral Gene Induction in Stable Cell Lines
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Lentiviral plasmid (pLVX-TetOne-Puro-EGFP, pLVX-TetOne-Puro-FLAG-RSL1D1, pLVX-TetOne-Puro-FLAG-RSL1D1-NT, pLVX-TetOne-Puro-FLAG-RSL1D1-CT, or pLenti6/V5-GW/p53), packaging plasmid pCMV-dR8.2, and envelop plasmid pCMV-VSV-G were co-transfected into Lenti-X™ 293 T cells (80 to 90% confluence) to produce lentiviruses using Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) according to the reagent manual. The lentiviruses were transduced into cells according to the Addgene pLKO.1 protocol (http://www.addgene.org/protocols/plko/ ). After transduction, cells were selected with 2 μg/mL puromycin (for pLVX-TetOne-Puro) or 10 μg/mL blasticidin S (for pLenti6/V5-GW/p53) for 5 days to establish stable cell strains, followed by treatment with 1 μg/mL puromycin or 10 μg/mL blasticidin S to maintain drug resistance. The stable cells were treated with 1 μg/mL doxycycline for 24 h (for RSL1D1-NT and RSL1D1-CT) or 72 h (for RSL1D1) to induce gene expression.
6
Cell Line Maintenance Protocols
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MDA-MB-231, HCC70 and T2 cell lines were from American Type Culture Collection. Lenti-X™ 293T cells were from Takara Bio (Takara Bio, San Jose, CA). The Lenti-X™ 293T cells, MDA-MB-231 and MSLN-MDA-MB-231, produced by lentiviral transduction, were maintained in DMEM with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.). HCC70 cells were cultured in RPMI-1640 with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). The T2 cell line was cultured in RPMI-1640 with 10% FBS and 2 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.). All cell lines were cultured at 37°C with 5% CO2.
7
Culturing Human Keratinocytes and Lenti-X 293T Cells
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Human keratinocyte cell lines, namely NIKS (ATCC CRL‐12191) and NIKS18 (NIKS cells contain HPV18 genome as episomes), were cultured in F medium with mitomycin C‐treated 3T3 mouse fibroblasts as previously described.
21 (link),
22 (link) The detail is shown in AppendixS1 . Lenti‐X 293T cells (Thermo Fisher Scientific) were cultured in DMEM (Thermo Fisher Scientific) with 10% FBS (Nichirei Biosciences Inc.).
21 (link),
22 (link) The detail is shown in Appendix
8
Lentiviral SMNDC1 Knockdown Protocol
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SMNDC1 knock-down was performed as described in Casteels et al.9 (link). Briefly, Smndc1 shRNA from the TRC shRNA library (https://portals.broadinstitute.org/gpp/public/ ) (TRCN0000123795) was cloned into pLKO.1 (Addgene plasmid #10878). This plasmid was packaged into lentivirus in Lenti-X™ 293 T cells (BOSC-23, RRID:CVCL_4401, TakaraBio Cat#632180) with Lipofectamine™ 3000 (Thermo Fisher Scientific L3000008) and packaging plasmids psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259). Target cells were transduced with viral supernatant after filtering and addition of 8 µg/ml Polybrene® (Santa Cruz Biotechnology sc-134220) 48 h after transfection. Medium was changed 24 h later.
9
Lentiviral Production from Tet-Fuw Plasmids
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Plasmid DNA of Tet-Fuw-OSKM (Addgene #20231), Tet-Fuw-Ascl1 (Addgene #27150), Tet-Fuw-M2rtTA (Addgene #20342), pMD2.G (Addgene #12259), and psPAX2 (Addgene #12260) were used for packaging lentivirus. Lenti-X™ 293T Cells (Clontech, Cat# 632180) were cultured in 10-cm petri dish with DMEM medium (Thermo Fisher SCIENTIFIC) plus 10% FBS and 1% Pen Strep (Thermo Fisher Scientific) at 37 °C with 5% CO2. For lentivirus production, 10 µg of Tet-Fuw-cDNA (OSKM, M2rtTA or Ascl1) with 2.5 µg of pMD2.G, 7.5 µg of psPAX2 were transfected into Lenti-X™ 293T Cells by using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Cat# L3000-015). Nine hours after initial transfection, cell culture medium was changed into DMEM medium supplemented with 5% heated-inactivated FBS. Pseudoviurs-containing culture medium was collected into sterile capped tubes 48 and 72 h post transfection, centrifuged at 500g for 10 min and filtered through 0.45 μm polyethersulfone (PES) filter membranes. The lentivirus medium was stored at -80 °C.
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