293ltv cells

Manufactured by Cell Biolabs

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293LTV cells are a human embryonic kidney cell line that has been modified to stably express the large T antigen from the SV40 virus. These cells provide a convenient system for producing recombinant proteins and for the generation of lentiviral vectors.

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HEK 293LTV cell line (7 citations) 293LTV cell line (2 citations)

10 protocols using 293ltv cells

Overexpression of CES2 in SU.86.86 Cancer Cells

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Parental SU.86.86 cells were purchased from ATCC. Manipulated SU.86.86 cell lines were provided by Dr Samir Hanash from MD Anderson Cancer Center and were cultured as previously described.11 (link) For overexpression, CES2 was cloned into pLenti-C-Myc-DDK-IRESPuro (OriGene, Rockville, MD) vector, an empty vector was used as a control. Lentiviral infections were performed using 293LTV cells (Cell Biolabs, Inc., San Diego, CA) as producers of viral supernatants. 293LTV cells were grown on 10 cm plates to 70% confluence and cotransfected with lentiviral DNA and the helper vectors pCMVΔ8.74 and pVSV-G (Clontech, Mountain View, CA) using Lipofectamine 3000 (Life Technologies), according to the manufacturer's instructions. The medium was harvested 48 h post-transfection, filtered through a 0.45 μm filter and used to cross-transduce cancer cells in the presence of 8 μg mL^–1 Polybrene (Sigma-Aldrich). Subsequently clones were selected by puromycin. All cells were maintained in a 5% CO₂ atmosphere at 37 °C. SU.86.86 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin/antimycotic (complete growth medium).

Kailass K., Sadovski O., Capello M., Kang Y., Fleming J.B., Hanash S.M, & Beharry A.A. (2019). Measuring human carboxylesterase 2 activity in pancreatic cancer patient-derived xenografts using a ratiometric fluorescent chemosensor †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc00283a. Chemical Science, 10(36), 8428-8437.

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Lentiviral Transduction and Knockdown

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cDNAs were cloned into pLX304 lentiviral expression vectors (Addgene) or pBabe (Addgene). pLKO.1 shRNA lentiviral vector construct designed by the RNAi Consortium were used as described previously (18 (link)). Lentivirus coding for cDNA or shRNA were packaged in 293LTV cells (Cell Biolabs) and transduced to the target cells as previously described (18 (link)). cDNA/shRNA/siRNA/sgRNA source, sequences and RNA depletion procedures are provided in the Supplementary Materials and Methods.

Becker J.H., Gao Y., Soucheray M., Pulido I., Kikuchi E., Rodríguez M.L., Gandhi R., Lafuente-Sanchis A., Aupí M., Fernández-Coronado J.A., Martín-Martorell P., Cremades A., Galbis-Caravajal J.M., Alcácer J., Christensen C.L., Simms P., Hess A., Asahina H., Kahle M.P., Al-Shahrour F., Borgia J.A., Lahoz A., Insa A., Juan O., Jänne P.A., Wong K.K., Carretero J, & Shimamura T. (2019). CXCR7 reactivates ERK signaling to promote resistance to EGFR kinase inhibitors in NSCLC.. Cancer research, 79(17), 4439-4452.

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Cell Line Authentication and Culture

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SW480, LS174T, WiDr, HCT116, and COLO320 cell lines were obtained from ATCC. 293LTV cells were from Cell Biolabs. All cell lines were routinely screened for Mycoplasma infection by PCR (once a month in general and before every animal experiment in particular). SW480 and HCT116 cells were cultured in McCoy’s 5A supplemented with 10% FBS, sodium pyruvate, and L-glutamine. LS174T and 293LTV cells were cultured in DMEM supplemented with 10% FBS, COLO320 cells were cultured in RPMI-1640 supplemented with 10% FBS, and WiDr cells were grown in EMEM supplemented with 10% FBS. All commonly used cell lines were routinely authenticated using STR profiling at UC Berkeley Sequencing Facility.

Yu J., Navickas A., Asgharian H., Culbertson B., Fish L., Garcia K., Olegario J.P., Dermit M., Dodel M., Hänisch B., Luo Y., Weinberg E.M., Dienstmann R., Warren R.S., Mardakheh F.K, & Goodarzi H. (2020). RBMS1 suppresses colon cancer metastasis through targeted stabilization of its mRNA regulon. Cancer discovery, 10(9), 1410-1423.

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Cell Line Culture Protocols for HIV Research

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The following cell lines were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID: U1, J1.1 and ACH-2 from Dr. Thomas Folks; J-Lat clone (6.3, 8.4, 9.2, 10.6 and 15.4), from Dr Eric Verdin, MT-4 [48] (link) from Dr. Douglas Richman, and were grown in R10 containing 50 U/mL of penicillin, 50 µg/mL of streptomycin, and 2 mM L-glutamine at 37°C and 5% CO₂ atmosphere. 293LTV cells [49] (link) were purchased from Cell Biolabs (USA) and were grown in DMEM (high glucose) (Life Technologies), 10% fetal bovine serum (FBS), 0.1 mM MEM Non-Essential Amino Acids, 2 mM L-glutamine, 50 U/mL penicillin, and 100 µg/mL streptomycin at 37°C and 5% CO₂ atmosphere.

Abreu C.M., Price S.L., Shirk E.N., Cunha R.D., Pianowski L.F., Clements J.E., Tanuri A, & Gama L. (2014). Dual Role of Novel Ingenol Derivatives from Euphorbia tirucalli in HIV Replication: Inhibition of De Novo Infection and Activation of Viral LTR. PLoS ONE, 9(5), e97257.

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Generating NRF2 Knockout Cell Lines

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NFE2L2 (NRF2) sgRNA crispr/cas9 all-in-one lentivector was purchased from Applied Biological Materials (Abm, Richmond, BC, Canada) and co-transfected with second generation packaging mix (Abm) into 293LTV cells (Cell Biolabs, San Diego, CA) following supplier’s protocol. Cells were infected with NRF2 lentiviral particles with polybrene at a concentration of 8 μg/ml. Cells were selected with puromycin and single clones were isolated. NRF2 mRNA and protein levels were examined using qRT-PCR and Western blotting.

Peng D., Lu H., Zhu S., Zhou Z., Hu T., Chen Z., Zaika A, & El-Rifai W. (2019). NRF2 antioxidant response protects against acidic bile salts-induced oxidative stress and DNA damage in esophageal cells. Cancer letters, 458, 46-55.

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Cell Culture Protocols for Common Cell Lines

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The human embryonic kidney cell line HEK293 [51 (link)] and the African green monkey cell line COS-7 [52 (link)] was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany). 293LTV cells were obtained from Cell Biolabs, San Diego, CA, USA. The human glioblastoma cell line LN-405 [53 (link)] was a kind gift from I. Schulz, Probiodrug AG, Halle, Germany. The human lung adenocarcinoma cell line A549 [54 (link)] was obtained from CLS, Eppelheim, Germany. HEK293 cells, LN-405 cells, and COS-7 cells were grown in DMEM (Life Technologies, Carlsbad, CA, USA), supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA). 293LTV cells were grown in DMEM, supplemented with 10% FBS, 1x GlutaMAX (Life Technologies, Carlsbad, CA, USA) and 1x non-essential amino acids (Life Technologies, Carlsbad, CA, USA). A549 cells were cultivated in RPMI1640 medium supplemented with 10% FBS. All cell lines were cultivated at 37 °C with 5% CO₂ (HEK293, 293LTV, COS-7) or 10% CO₂ (LN-405).

Gröger V., Wieland L., Naumann M., Meinecke A.C., Meinhardt B., Rossner S., Ihling C., Emmer A., Staege M.S, & Cynis H. (2020). Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level. International Journal of Molecular Sciences, 21(21), 7855.

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Generating Lentiviral Particles for CRISPR

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To generate lentivirus, the human lentiCRISPRv2 plasmid library (Addgene 1000000048) was co-transfected with packaging plasmids pCAG-VSVG and psPAX2 (Addgene plasmids 35616 and 12260, respectively). Briefly, a T-75 flask of 80% confluent 293LTV cells (Cell Biolabs) was transfected in OptiMEM (Thermo Fisher Scientific) using 8 μg of the lentiCRISPRv2 plasmid library, 4 μg pCAG-VSVG, 8 μg psPAX2, 2.5 μg pAdVantage (Promega), 30 μl of P3000 Reagent (Thermo Fisher Scientific), and 30 μl of Lipofectamine 3000 (Thermo Fisher Scientific). Cells were incubated overnight and then media was changed to DMEM (Sigma-Aldrich) with 10% BCS and 1x GlutaMAX (Thermo Fisher Scientific). After 48 h, viral supernatants were collected and centrifuged at 2000 rpm for 10 min to get rid of cell debris. The supernatant was filtered through a 0.45μm ultra-low protein binding filter (Merck Millipore). Aliquots were stored at −80 °C.

Lau M.T., Manion J., Littleboy J.B., Oyston L., Khuong T.M., Wang Q.P., Nguyen D.T., Hesselson D., Seymour J.E, & Neely G.G. (2019). Molecular dissection of box jellyfish venom cytotoxicity highlights an effective venom antidote. Nature Communications, 10, 1655.

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Pancreatic Cancer Cell Line Characterization and Manipulation

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Pancreatic cancer cell lines used in this study (BxPC-3 [#CRL-1687), SW1990 [#CRL-2172], SU.86.86 [#CRL-1837], PANC-1 [#CRL-1469], Hs 766T [#HTB-134], CFPAC-1 [#CRL-1918], MIA PaCa-2 [#CRM-CRL-1420], AsPC-1 [#CRL-1682], Panc 03.27 [#CRL-2549], HPAF-II [#CRL-1997], and Capan-2 [#HTB-80]) were purchased from ATCC and maintained in RPMI-1640 with 10% fetal bovine serum (FBS). The identity of each cell line was confirmed by DNA fingerprinting via short tandem repeats at the time of mRNA and total protein lysate preparation using the PowerPlex 1.2 kit (Promega). Fingerprinting results were compared with reference fingerprints maintained by the primary source of the cell line.
Transient knockdown of CES2 was performed by transfecting the cells using the following siRNAs: siControl (Silencer Select Negative Control No. 1, Thermo Fisher Scientific) and siCES2 (s225041, s528; Thermo Fisher Scientific). Short-hairpin RNAs targeting human CES2 mRNA and cloned into the pLKO.1-puro vector were obtained from the human library MISSION® TRC-Hs 1.0 (Sigma–Aldrich, TRCN0000046965). For overexpression, CES2 was cloned into the pLenti-C-Myc-DDK-IRESPuro (OriGene) vector, and an empty vector was used as a control. Lentiviral infections were conducted using 293LTV cells (Cell Biolabs, Inc.).

Chen Y., Capello M., Rios Perez M.V., Vykoukal J.V., Roife D., Kang Y., Prakash L.R., Katayama H., Irajizad E., Fleury A., Ferri-Borgogno S., Baluya D.L., Dennison J.B., Do K.A., Fiehn O., Maitra A., Wang H., Chiao P.J., Katz M.H., Fleming J.B., Hanash S.M, & Fahrmann J.F. (2021). CES2 sustains HNF4α expression to promote pancreatic adenocarcinoma progression through an epoxide hydrolase-dependent regulatory loop. Molecular Metabolism, 56, 101426.

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Breast Cancer Cell Line Manipulation

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The MDA-MB-231 (MDA-parental, ATCC HTB-26 (link)) human breast cancer cell line, its highly metastatic derivative, MDA-LM2 (57 (link)), and 293LTV cells (Cell BioLabs LTV-100) were cultured in DMEM medium supplemented with 10% FBS, penicillin, streptomycin and amphotericin. The HCC1806-LM2 cell line (an in vivo selected highly lung metastatic derivative of the HCC1806 breast cancer line (ATCC CRL-2335)) was cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin, streptomycin and amphotericin. Gene knockdowns were performed using stably expressed shRNA constructs or siRNA transfection. Gene overexpression was performed using stably expressed ORFs. Splicing inhibition was performed by morpholino transfection. Endogenous S3Es were deleted by transfecting gRNA-Cas9 RNPs (IDT), programmed with gRNAs flanking S3E regions.

Fish L., Khoroshkin M., Navickas A., Garcia K., Culbertson B., Hänisch B., Zhang S., Nguyen H.C., Soto L.M., Dermit M., Mardakheh F.K., Molina H., Alarcón C., Najafabadi H.S, & Goodarzi H. (2021). A pro-metastatic splicing program regulated by SNRPA1 interactions with structured RNA elements. Science (New York, N.Y.), 372(6543), eabc7531.

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Lentiviral Knockdown and Overexpression of APE1

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APE1 shRNA and control shRNA lentiviral plasmids were obtained from Vector Builder Inc. (Santa Clara, CA, USA). Plasmids were co-transfected with second generation packaging mix (Abm) into 293LTV cells (CellBiolabs, San Diego, CA), following the manufacturer's protocol. After 72 h transfection, the media supernatants were collected and stored at −80 °C. CPB, FLO1 and OE33 cells were infected with control or APE1shRNA media supernatants in the presence of polybrene (4 μg/mL) for 72 h. Stable cell lines expressing control or APE1shRNA were selected using puromycin and used for further experiment. The FLAG-tagged coding sequence of APE1 and its mutant C65A were cloned into pcDNA3.1 mammalian expression plasmid (Invitrogen, Carlsbad, CA USA).

Sriramajayam K., Peng D., Lu H., Zhou S., Bhat N., McDonald O.G., Que J., Zaika A, & El-Rifai W. (2021). Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells. Redox Biology, 43, 101970.

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