According to the manufacturer' s instructions, Overexpression Lentivirus was produced via the co-transfection of 293T cells with a pEZ-lv105 vector (pEZ-Mock or pEZ-TNFRSF11B) and lentiviral packaging mix (Invitrogen, Carlsbad, CA,USA). According to the manufacturer's instructions, Knockdown Lentivirus was produced by the co-transfection of 293T cells with a piLenti vector (pEZ-Scramble or pEZ-shTNFRSF11B) and lentiviral packaging mix (Invitrogen, Carlsbad, CA, USA). After transfection for 48 hours, Lentivirus-containing supernatant was collected, centrifuged, and stored at -80°C. For transfection of the virus, 1 mL of the Mock or TNFRSF11B lentiviruses was incubated with HGC- 27 and BGC-823 cells overnight at 37°C in a 5% CO2 humidified cell culture incubator. Stable GC cells with increased endogenous TNFRSF11B expression were selected via culturing in puromycin (1 μg/ml).1 mL of the Scramble or shTNFRSF11B lentiviruses was incubated with MGC-803, SGC-7901 cells overnight at 37°C in a 5% CO2 humidified cell culture incubator. Stable GC cells with decreased endogenous TNFRSF11B expression was selected via culturing in puromycin (1 μg/ml).
Lentiviral packaging mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Lentiviral Packaging Mix is a pre-formulated set of plasmids designed for the production of lentiviral particles. It contains the essential viral components required for the generation of replication-incompetent lentiviral vectors.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
293ft cell, by Thermo Fisher Scientific (1 mentions)
Plv final puro ef1α apoptin, by Thermo Fisher Scientific (1 mentions)
Millex hv syringe filter, by Merck Group (1 mentions)
Viralpower lentiviral expression system, by Thermo Fisher Scientific (1 mentions)
Plenti6 v5 gw lacz, by Thermo Fisher Scientific (1 mentions)
11 protocols using lentiviral packaging mix
1
Lentivirus-Mediated TNFRSF11B Modulation in Gastric Cancer
2
Lentiviral Vector-Based Stable Cell Lines
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Stable cell lines were established with a lentiviral vector using previously described protocols22 (link). Briefly, lentivirus was produced by the co-transfection of HEK293FT cells with lentivirus expression vectors and lentiviral packaging mix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Specifically, packaging plasmids were co-transfected into HEK293FT cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) with short hairpin RNA constructs for targeting ISL1. ISL1-specific shRNA oligos are listed in Supplementary Table 1 . Identification of stable cell lines was performed using RT-PCR and western blotting for quantifying the expression levels of ISL1. All the primers are provided in Supplementary Table 2 .
3
GSN Knockdown Using Lentiviral RNAi
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Three pairs of short hairpin RNAs were designed and synthesized according to GSN CDS sequences and then reverse transcribed to DNA. The DNA sequences were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid vector (R&S Biotechnology, China). The cloned fragments were amplified by PCR and subcloned into the pLenti6.3/V5-DEST vector (R&S Biotechnology). The lentivirus vector plasmid and lentiviral packaging mix (Invitrogen, Thermo Fisher Scientific, Inc., USA) were transiently co-transfected into 293T cells (R&S Biotechnology). Recombinant lentivirus-infected HPDE6-C7 cells with the highest degree of silencing were screened by qRT-PCR (18 (link)).
4
Lentiviral Knockdown of NuMA in MDCK Cells
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The GFP coding sequence from pLV-mU6-EF1-GFP (Biosettia) vector was replaced with H2B-mCherry to generate pLV-mU6-EF1-H2B-mCherry. Lentivirus-mediated stable knockdown of NuMA in MDCK cells was carried out as previously described (Zheng et al., 2010 (link); Wan et al., 2012 (link)). Briefly, long oligos containing control and target sequences were cloned downstream of the U6 promoter in pLV-mU6-EF1-H2B-mCherry to generate specific shRNA vectors. Once sequence was verified and knockdown efficiency tested by transient transfection, the shRNA vectors were cotransfected with the lentiviral packaging mix (Invitrogen) into HEK293FT cells, and the pseudovirus containing supernatant was collected 48 h posttransfection. Virus supernatant was used to infect MDCK cells cultured in 12-well plates. At 24 h after infection, the cells were passaged onto P-100 plates, and transduced clones (based on virus-mediated expression of H2B-mCherry) were marked and isolated using cloning rings 1 wk later. The knockdown efficiency was analyzed by Western blot and immunostaining. Target sequences for canine NuMA were 5′-GCTTTCAGCATCCTCAATACA-3′ and 5′-GCTTGCGGATGAGAGAAATAA-3′.
5
Lentiviral Transduction of Apoptin in MSCs
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Lentiviral particles carrying apoptin gene were prepared by transient co-transduction of pLV/Final-puro-EF1α-apoptin (Invitrogen Life Technologies) and a lentiviral packaging mix (Invitrogen Life Technologies) into 293FT cells (Invitrogen Life Technologies) using Lipofectamine 2000 (Invitrogen Life Technologies), according to manufacturer’s instructions. At 48 h after transfection, viral particles were harvested, filtered through a 0.45-μm polyethersulfone membrane and concentrated by ultracentrifugation at 50,000 × g for 1 h at 4°C. Titers of concentrated lentivirus particles changed from 4×107 to 9×107 U/ml. Human MSCs were transduced with lentiviral particles at an infection multiplicity of 50. Following two rounds of infection, puromycin (HyClone Laboratories, Inc.) was added to the culture medium at a concentration of 1–5 μg/ml and maintained for 2–3 days. The obtained MSC lines were defined as MSCs APOPTIN. At the same time, lentiviral particles carrying hrGFP were prepared with pLV/Final-puro-EF1α-hrGFP (Invitrogen Life Technologies) and transduced into MSCs in parallel to assess the modification efficiency of apoptin.
6
MELK Knockdown Protocol for Gastric Cancer Cells
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The MELK expression knockdown procedure was conducted as previously described [35 (link)]. Lentivirus was produced by the co-transfection of 293T cells with a pLenti vector (pGLV3-shControl or pGLV3-shMELK) and lentiviral packaging mix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Lentivirus-containing supernatant was harvested at 48 hours post-transfection, centrifuged, and stored at −80°C. For viral transductions, 1 ml of the scrambled shControl or shMELK lentiviruses was incubated with BGC823 and SGC7901 cells overnight at 37°C in a humidified cell culture incubator. Stable GC cells with depleted endogenous MELK expression were selected by culturing in puromycin (0.8 μg/ml).
7
Lentiviral Transduction of Cell Lines
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The lentiviral particles carrying the IFN-γ or humanized Renilla GFP genes were prepared by transient cotransfection of pLV/Final-puro-EF1α-IFN-γ or pLV/Final-puro-EF1α-hrGFP with a lentiviral packaging mix (Invitrogen) into 293FT cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hours, the viral particles were harvested, filtered through a 0.45 μm polyethersulfone membrane, and concentrated by ultracentrifugation. Human MSCs were transduced with the lentiviral particles carrying IFN-γ or hrGFP at a multiplicity of infection of 50. At the same time, the 3T3, 293FT, H460, H1299, A549, and MCF-7 cell lines were transduced with lentiviral particles carrying only hrGFP using the same protocol. After two rounds of infection, 1–5 μg/mL puromycin was added to the culture medium, and these cultures were maintained for 2-3 days. The isolated cell lines were defined as MSCs IFN-γ, MSCs hrGFP, 3T3 hrGFP, 293FT hrGFP, H460 hrGFP, H1299 hrGFP, A549 hrGFP, and MCF-7 hrGFP, respectively.
8
ASPH Gene Knockout Using CRISPR-Cas9
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In the BGC823 and SGC7901 cell lines, the ASPH gene was knocked out using CRISPR–Cas9 targeting ASPH. According to the manufacturer’s instructions, lentivirus was produced by means of co-transfection of lentiviral-packaging mix (Invitrogen, Carlsbad, CA, USA) and NTC (non-target control, LentiCRISPR_V2) or sgASPH into HEK293FT cells, using Lipofectamine 2000 (Life Technologies). The detailed sequence of the ASPH sgRNA was as follows: 5′-ATGGAGGACACAAGAAT-3′. After transfection for 48 h, stable ASPH knockout cells were selected with 1.0 μg/mL puromycin for 7 days, and ASPH knockout cell clones were screened by Western blotting.
9
CRISPR-Mediated TfR1 Knockout in Gastric Cancer Cells
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TfR1-knockout expression was established by CRISPR–Cas9 targeting TfR1 in BGC823 and SGC7901 cell lines. Lentivirus was produced by co-transfection of HEK293FT cells with LentiCRISPRv2, LentiCRISPRv2_sgTfR1-1, and LentiCRISPRv2_sgTfR1-2, respectively, together with lentiviral-packaging mix (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction, which was transfected with Lipofectamine 2000 (Life Technologies). For the detailed sequences of the sgRNA: sgTfR1-1: 5′-CACCCG CTA TAC GCC ACA TAA CCC CC-3′; sgTfR1-2: 5′-CACCCG CTG CAG CAC GTC GCT TAT AT-3′. Stable TfR1-knockout cells were selected with puromycin (1.0 mg/ml) for 14 days after transfection for 48 h, and successfully TfR1-knockout cell clones were identified by flow cytometry.
10
Lentiviral Expression of NOX4 Protein
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The NOX4/pCMV-SPORT6 vector was obtained from 21C Frontier Human Gene Bank (KRIBB, Daejeon, Korea). Lentivirus was produced using the Viralpower Lentiviral Expression System (Invitrogen). Briefly, the NOX4 open reading frame (ORF) was amplified from the NOX4/pCMV-SPORT6 vector using the following primers: forward (5′-CCCGGGACCATGGCTGTGTCC-3′) and reverse (5′-GCGGCCGCTCAGCTGAAAGACTC-3′). The NOX4 cDNA was subcloned into the pLenti6/V5-D-TOPO vector according to the manufacturer's protocol. The resultant NOX4/pLenti6-D-TOPO vector was verified using PCR, restriction digestion, and sequence analysis (Macrogen, Seoul, Korea). The pLenti6/V5-GW/lacZ (Invitrogen) vector was used as a positive control. Recombinant lentiviruses were produced according to the manufacturer's protocol (Invitrogen). Briefly, we co-transfected the pLenti6/V5-D-TOPO (NOX4 or LacZ) vector and the Lentiviral Packaging Mix (Invitrogen) into 293 FT cells. After 48 h, the viral supernatant was collected, spun to remove cell debris, filtered through a Millex-HV syringe filter (0.45 μm, Millipore, Bedford, MA, USA), and stored at −80 °C.
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