The human HCC cell lines Hep3B and Huh-7 were obtained from the American Type Culture Collection (Manassas, VA) and the Japanese Collection of Research Bioresources (Tokyo, Japan), respectively. GP2-293 cells were purchased from Clontech (BD Biosciences, San Jose, CA). SMMC-7721 cells were purchased from the China Center for Type Culture Collection (Wuhan, China). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), containing 10% fetal bovine serum and 1% antibiotic-antimycotic (Beyotime, Haimen, China), at 37°C with 5% CO2. For all studies, vesicle-depleted medium was prepared by centrifuging cell-culture medium at 110,000g overnight to spin down any preexisting vesicular content.9 (link)
Gp2 293 cells
Manufactured by Takara Bio
Sourced in United States, Japan
GP2-293 cells are a human embryonic kidney cell line commonly used in cell and gene therapy research. They are engineered to stably express the gammaretroviral gag and pol genes, which facilitates the production of high-titer, replication-incompetent retroviral vectors. The GP2-293 cell line serves as a reliable platform for the generation of recombinant retroviruses.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Pvsv g, by Takara Bio (4 mentions)
Polybrene, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
X tremegene 9 dna transfection reagent, by Roche (2 mentions)
Transit lt1, by Mirus Bio (2 mentions)
Incucyte live cell analysis system, by Sartorius (2 mentions)
Gp2 293, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
0.45 μm pore size filter, by Merck Group (2 mentions)
293ft cell, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Plncx2 retroviral vector, by Takara Bio (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Vsv g vector, by Takara Bio (2 mentions)
Fetal bovine serum (fbs), by Beyotime (1 mentions)
41 protocols using gp2 293 cells
1
Cell Culture Techniques for Hepatocellular Carcinoma
2
Murine Cytokine Expression Vectors
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The precise cDNA sequence of murine IL-18 or GM-CSF followed by that of the B7 transmembrane domain was subcloned into the retroviral vector pLNCX (BD Biosciences, San Diego, USA) using a standard procedure (Fig 1B ). Recombinant retroviral particles were packaged by co-transfection of pVSVG with pLNCX constructs into GP2-293 cells (Clontech, USA). After 48 hours, the harvested culture medium was filtered through a 0.22-μm syringe filter, followed by mixing with polybrene to 8 μg/ml. It was then added to CT26 colon carcinoma cells for virus infection. The stable CT26 cells were selected by G418 and were sorted by FACScaliber flow cytometer to establish CT26/IL-18, CT26/GM-CSF and CT26/GM-CSF/IL-18 clones.
3
Rab8b Knockdown in Mouse MEFs
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Rab8b-specific lentiviral shRNA construct targeting against the 3'UTR of mouse Rab8b was constructed by inserting the annealed complementary oligonucleotides (5'-CCGGGCCAAGAACTAACAGAACTTTCCATGGAAAGTTCTGTTAGTTCTTGGCTTTTTG-3' and 5'-AATTCAAAAAGCCAAGAACTAACAGAACTTTCCATGGAAAGTTCTGTTAGTTCTTGGC-3') into pLK0.1 lentiviral vector (Addgene) between AgeI and EcoRI sites. For viral packaging, this lentiviral vector along with pVSV-G was transfected into GP2-293 cells (Clontech) using Lipofectamine 2000 (Invitrogen). After 48 h, the supernatant was collected and subjected to ultracentrifugation (15,000g, 2 h) for viral concentration. The viral pellet was resuspended with 200 μl of DMEM and aliquoted for later usage. For Rab8b KD, MEFs were infected with diluted lentivirus stock (1:50,000) for 5 h in DMEM in the presence of polybrene (8 μg/ml) and then incubated with complete DMEM containing 10% FBS for another 24 h. Puromycin (3 μg/ml) was added into the culture medium for selection. KD efficiency and the maintenance of KD were confirmed by Western blotting.
4
Tumor-Astrocyte Co-culture Assay for Therapeutics
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GP2-293 cells (Catalog #. 631458) was obtained from CLONTECH, now TAKARA BIO USA (Mountain View, CA). Rat C6 GBM (Catalog# CCL-107), and mouse NIH3T3 fibroblasts (Catalog# CRL-1658) were obtained from ATCC (Manassas, VA). Human U251 GBM (Catalog# 03063001) was from SIGMA-ALDRICH (St. Louis, MO). All cells were maintained following manufacturer’s protocols. Normal human astrocytes (HA, Catalog# CC-2565) were purchased from LONZA (Basel, Switzerland) and grown in HA growth medium kit (Catalog# 821–500) from APPLICATION INC. (San Diego, CA). For the co-culture system experiments, labeled tumor and unlabeled non-tumor cells were seeded in 6-well plates at designated densities and treated with BPM31510 or vehicle, for 24–72 h. Rat C6 GBM and NIH3T3 fibroblast cells were co-cultured in DMEM medium supplemented with 10% FBS, and human U251 GBM and HA cells were co-cultured in HA growth medium.
5
Generation of K-Ras Oncogene Expressing Cells
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K-RasG12V cDNA was prepared from pGCDN-K-RasG12V-IRES-Kusabira Orange [34 (link)], and was cloned into the retroviral vector pMXs-IRES-GFP. GP2-293 cells (Clontech) were transfected with the resulting vectors and the VSV-G envelope plasmid with the use of the FuGene HD reagent (Promega) and were cultured for 48 or 72 h, after which the retrovirus-containing culture supernatants were harvested. The CRISPR-Cas9 knockout plasmid and HDR Plasmid for Trp53 were obtained from Santa Cruz Biotechnology.
6
Generating Recombinant Retroviral Particles
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Individual coding DNA fragments, including PF (1–515 amino acids), peptide substrate of PSA (GPLGVR), peptide substrate of MMP-2 (HSSKLQ), a granzyme B cDNA and a CEBPD cDNA, were cloned into a pLHCX vector. Later, the expression vectors were co-transfected with pVSVG expression vector into GP2-293 cells (Clontech, Mountain View, CA, USA) to produce recombinant retroviral particles for the infection of 3T3 cells and to generate stable cell lines expressing PSA-specific fusion proteins or MMP-specific fusion proteins.
7
Retroviral Transduction of EL4 Cells
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To transduce EL4 cells, retrovirus pseudotyped with the vesicular stomatitis virus (VSV-G) envelope was produced by polyethylenimine (PEI, molecular weight 4000, PolySciences Catalogue No 24885–2, Warrington, PA, USA) transfection of GP2-293 cells (Clontech, Palo Alto, CA, USA). NaOH-neutralised PEI (1 mg/ml) was complexed with 6.8 µg of rMSCV-mCCL3-mScarletI and rMSCDV-mCCL4-miRFP670 and 3.2 µg of pMD2.G plasmid (VSVG coding sequence expressed from the CMV promoter, kind gift of Didier Trono) for 30 min at room temperature before addition to 7 × 106 GP2-293 cells. At 72 hr after transfection, viral supernatant was used to transduce EL4 cells and fluorescent EL4 cells were sorted (BD FACS Aria III) 72 hr after transduction. GP2-293 cells were maintained in DMEM (Gibco) containing 4.5 g/L glucose, 4 mM L-glutamine and 1 mM sodium pyruvate supplemented with 10% heat-inactivated FBS. Sorted CCL3/CCL4-secreting or mTagBFP2-expressing EL4 tumour cells were cultured in TCM with routine passaging three times per week, maintained at cell densities under 1 × 106/ml.
8
Rhesus Macaque MHC Class I Transduction
9
Generating Lentivirus and Tet-on HEK293 Cells
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To make lentivirus, control or AGO2 shRNA plasmids or 3 × Flag-hAGO2 plasmids were co-transfected into 293T cells (ATCC, CRL-3216) with pΔ8.9 and pVSV-G in ratio 4:3:2. pTet-on Advanced and pMD2.G vectors were co-transfected into GP2-293 cells (ClonTech, 631458) in ratio 1:1 to produce Tet-on retrovirus. All viruses were collected at 48 h after transfection. To produce Tet-on HEK293 cells, HEK293 cells (ATCC, CRL-1573) were infected by the Tet-on retrovirus for 48 h followed by 500 μg per ml G418 selection. Then HEK293 cells were infected by the control or AGO2 shRNA lentivirus, and HEK293-Tet-on cells were infected by the control or 3 × Flag-hAGO2 lentivirus for 48 h followed by 2 μg per ml puromycin selection.
HEK293 cells were cultured with DMEM medium containing 10% FBS. HEK293 cells with AGO2 knockdown were cultured with DMEM medium containing 10% FBS and 0.5 μg per ml puromycin. HEK293 cells with AGO2 overexpression were cultured with DMEM medium cantaining 10% FBS, 0.5 μg per ml puromycin and 100 μg per ml G418.
HEK293 cells were cultured with DMEM medium containing 10% FBS. HEK293 cells with AGO2 knockdown were cultured with DMEM medium containing 10% FBS and 0.5 μg per ml puromycin. HEK293 cells with AGO2 overexpression were cultured with DMEM medium cantaining 10% FBS, 0.5 μg per ml puromycin and 100 μg per ml G418.
10
Production of VSV-G Pseudotyped Retrovirus
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To produce VSV-G pseudotyped retrovirus, 4 × 106 of GP2-293 cells (Clontech Laboratories, Inc., Mountain View, CA, USA) were cultured in a 10 cm dish for 24 h, then co-transfected with plasmids pRetroX-IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA) and pVSV-G (Clontech Laboratories, Inc., Mountain View, CA, USA) by using X-tremeGENE 9 DNA Transfection Reagent (Roche, Indianapolis, IN, USA.). Cells were washed with PBS after 24 h and the virus-containing culture supernatant was harvested after 48 h. After centrifugation to remove residual cells, the virus containing supernatant was aliquoted and kept in an −80 °C freezer.
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