HEK293T cells (ATCC CRL-3216) were cultured in DMEM (Gibco 31966-021, Waltham, MA, USA) with 10% FBS and plated in 10 cm dishes. Cells were allowed to become ~90% confluent prior to transfection. A total of 5 μg of pLenti-C-Myc-DDK-PKD1L1 WT or variant was transfected per dish using the Lenti-vpak Lentiviral Packaging Kit (OriGene TR30037), following the manufacturer’s instructions. After 18 h, the culture medium was exchanged, and at 48 and 72 h post-transfection, the medium was collected and centrifuged at 600× g for 2 min. The supernatant was filtered through a 0.45 μm filter, and aliquots were stored at −80 °C until required.
Lenti vpak lentiviral packaging kit
Manufactured by OriGene
Sourced in United States
The Lenti-vpak Lentiviral Packaging Kit is a product designed for the production of lentiviral particles. It contains the necessary components to package lentiviral vectors, including plasmids encoding the viral structural proteins and packaging system.
Automatically generated - may contain errors
Lab products found in correlation
Lentiviral orf control clone, by OriGene (2 mentions)
Mission short hairpin rna shrna lentiviral transduction particles, by Merck Group (2 mentions)
Mission turbogfp control transduction particles, by Merck Group (2 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Pgfp c shlenti, by OriGene (1 mentions)
Pgfp c scr shlenti, by OriGene (1 mentions)
Bfgf2, by Thermo Fisher Scientific (1 mentions)
Ps100093, by OriGene (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Sineg, by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Megatran 1.0 transfection reagent, by OriGene (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
10 protocols using lenti vpak lentiviral packaging kit
1
Lentiviral Transduction of HEK293T Cells
2
Investigating Ku86 Knockdown in Rad52 B Cells
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The Ku86-specific shRNA lentiviral construct pGFP-C-Ku86-shRNALenti (TL502435) and non-effective 29-mer scrambled shRNA lentiviral construct pGFP-C-scr-shLenti (TR30021) were obtained from Origene Technologies. To generate the lentivirus, pGFP-C-shLenti vector and packaging vectors were used to cotransfect HEK293T cells according to the manufacturer's instructions (Lenti-vpak Lentiviral Packaging Kit, Origene). Viral supernatants were collected and used to transduce Rad52+/+ and Rad52−/− B cells. Transduced B cells were then stimulated with LPS plus IL-4 for 96 h before analysing GFP+ and IgG1+ B cells by flow cytometry—dead (7-AAD+) cells were excluded from analysis. Expressions of Rad52, Ku86 and β-Actin proteins in the transduced B cells were analysed by immunoblotting.
3
Lentiviral Transduction for Chondrogenesis
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Lentiviral production was performed in HEK293T cells using the Lenti-vpak Lentiviral Packaging Kit (Origene Technologies, Rockville, MD 20850, USA). In short, HEK 293 T cells were expanded in DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands), supplemented with 10% foetal calf serum (FCS; Life Technologies Europe BV, Bleiswijk, The Netherlands) 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and lentivirus particles were collected and titrated.
Following their isolation, hPACs were transduced at passage 1 with either control (pLV.CMV.bc.eGFP) or TNFRSF11B Lentivirus (MOI of 1). After 16 h, the medium was refreshed [DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 10% FCS (Life Technologies Europe BV, Bleiswijk, The Netherlands), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and 0.5 ng/ml bFGF-2 (PeproTech, London, UK)] and hPACs were further cultured for another passage. Subsequently, neo-cartilage was generated from 250 000 transduced hPACs in 3 D pellets for seven days, as described before [12 (link)], and keeping the conditions between both groups equal. All data were analysed 7 days following the 3D chondrogenesis.
Following their isolation, hPACs were transduced at passage 1 with either control (pLV.CMV.bc.eGFP) or TNFRSF11B Lentivirus (MOI of 1). After 16 h, the medium was refreshed [DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 10% FCS (Life Technologies Europe BV, Bleiswijk, The Netherlands), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and 0.5 ng/ml bFGF-2 (PeproTech, London, UK)] and hPACs were further cultured for another passage. Subsequently, neo-cartilage was generated from 250 000 transduced hPACs in 3 D pellets for seven days, as described before [12 (link)], and keeping the conditions between both groups equal. All data were analysed 7 days following the 3D chondrogenesis.
4
Lentiviral Overexpression and Knockdown of CD49b
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Lenti cDNA clone of human CD49b (RC212617L1) and Lenti-Vpak lentiviral Packaging Kit (TR30022) were purchased from Origene (Rockville, MD). The lentiviral particles were produced by transfecting the Lenti plasmid into HEK293T cells. The lentiviral particles were then tranduced into target cells and CD49b expression was examined using Western blotting. A CD49b human shRNA plasmid kit (TG312097) was purchased from Origene (Rockville, MD). CD49b shRNA plasmid or control plasmid was introduced into HOS or MG63 cells with Turbofectin 8.0 using manufacturer’s protocols for transient transfection followed by colony selection after treatment with 1 μg/ml puromycin.
5
Lentiviral Transduction of GFP-Tagged Proteins
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Lentiviral expression vectors with CMV promoters driving GFP-tagged human Sam68 and FADD were purchased from Origene (PS100093, RC200263L4, RC201805L4). Lentiviral particles containing these vectors were isolated using the Lenti-vpak Lentiviral Packaging Kit (Origene, TR30037) as directed by the manufacturer. When ready to transduce, lentivirus was thawed rapidly at 37 °C. Cells were seeded 50,000 cells in 1 mL maintenance media without any antibiotics into 6 well plates and reverse transduced with 500 μL of virus per well. Control wells were seeded in the absence of virus. After 48 h, cell lines were selected with puromycin at the following doses: MCF-10A .4 μg/mL for selection and maintenance, all cells grown in DMEM were selected at 2 μg/mL and maintained at 0.5 μg/mL, and all cells grown in RPMI were selected at 0.5 μg/mL and maintained at 0.25 μg/mL. After selection, cells were then flow-sorted at the Vanderbilt Flow Cytometry Shared Resource on the FACSAria III (BD) for the top 1% of GFP expression from the baseline cell pools and utilized in indicated experiments.
6
Stable MDCK Cell Lines with α-Catulin Knockdown
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MDCK cells were seeded on 6-well plate in density of 70,000 cells and the next day were transfected with specific small interfering (si)RNA targeting α-catulin gene (siCTNNAL1) (HSS112822, Thermo Fisher Scientific) or with universal negative control siRNA (siNeg) (#12935200, Thermo Fisher Scientific) using Opti-MEM medium (Thermo Fisher Scientific, #11058021) and Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, #13778100) according to manufacturer’s instructions. Before further experiments cells were cultured for 48 h. Effectiveness of silencing of α-catulin gene was confirmed by RT-qPCR. Independent MDCK cell lines were generated using lentiviral shRNA specific to canine α-catulin (KD1 and KD2) and non-effective shRNA scrambled cassette (control). Lentiviral cassettes were designed and cloned by OriGene (pGFP-C-shLentiviral vector, TR30023 and pGFP-C-shLenti-scrambled TR30021). Lentivirus was produced using Lenti-vpak Lentiviral Packaging Kit (Origene) according to the manufacturer protocol. GFP positive cells for stable cell lines were selected by FACS sorting.
7
Lentiviral Vectors for ORAI3 Modulation
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GFP-expressing lentiviral vectors expressing ORAI3-targeting shRNA (pGFP-lenti-Sh-ORAI3; OriGene: TL307587) and full-length ORAI3-expressing pCMV6-AC lentiviral vectors (OriGene: RC202325L1 NM_152288), respectively were packaged into HEK293T cells using the lenti-vpak lentiviral packaging kit (OriGene TR30037). Scrambled control shRNA and pCMV6-AC empty vector were used as negative controls. Jurkat cell lines stably expressing sh-ORAI3 or ORAI3 were established as per the manufacturer’s instructions (OriGene).
8
Lentiviral Transduction of TMEM47 in HCC
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The lentiviral open reading frame (ORF) clone of monomeric green fluorescent protein (GFP)-tagged human TMEM47 (cat. no. RC227689L2V) and lentiviral ORF control clone were purchased from OriGene Technologies, Inc. Lentiviral particles were produced by transfection into 293 cells using the Lentivpak Lentiviral Packaging kit (OriGene Technologies, Inc.). Suppression of TMEM47 was conducted by TMEM47-specific Mission® short hairpin RNA (shRNA) lentiviral transduction particles (Sigma-Aldrich; Merck KGaA). MISSION® TurboGFP control transduction particles (Sigma-Aldrich; Merck KGaA) was used as a control. Lentiviral particles were transduced into HCC cells according to the manufacturer's instructions. The expression levels of TMEM47 mRNA in HCC clones was examined by reverse transcription-quantitative PCR (RT-qPCR) analysis.
9
Lentiviral Transduction of ATM Variants
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ATM 3-52, ATM 4-53, ATM SINT and miniATM cDNAs (the sequences of the tested variants are reported in Fig. S1) were inserted into pLenti-C-Myc-DDK-IRES-neo tagged cloning vector with double selection: chloramphenicol for E. coli selection and neomycin for mammalian cell selection. WT hT and AT 648 hT untransduced cells were used as reference and negative control, respectively. Viral particles were produced by co-transfecting HEK 293T cells in 24-well plates (1.2 × 105/well) with cloned ATM variants using MegaTran1.0 Transfection Reagent as reported by the Lenti-vpak Lentiviral Packaging Kit (OriGene). Viral particles were collected and concentrated according to the method reported by Miller et al. [26 (link)].
10
Lentiviral Transduction of GSTA2 in HCC Cell Lines
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The human normal liver cell line named MIHA and the Hep3B hepatoma cell line were purchased from American Type Cell Culture (ATCC). The metastatic HCC cell line MHCC97L (97L in short) provided by the Liver Cancer Institute & Zhongshan Hospital of Fudan University, Shanghai, People's of Republic of China (27) . The cell lines were cultured in DMEM high glucose (Gibco; Thermo Fisher Scienti c, Inc.) with 10% FBS (Gibco; Thermo Fisher Scienti c, Inc.), 1% penicillium and streptomycin in a 37 ˚C incubator supplied with 5% CO 2 .
The lentiviral open reading frame (ORF) clone of monomeric green uorescent protein (GFP)-tagged human GSTA2 (cat. no. RC202479L2) and lentiviral ORF control clone were purchased from OriGene Technologies, Inc. Lentiviral particles were produced by transfection into HEK293 cells using Lenti-vpak Lentiviral Packaging kit (OriGene Technologies, Inc). Suppression of GSTA2 was conducted by GSTA2-speci c Mission® short hairpin RNA (shRNA) lentiviral transduction particles (Sigma-Aldrich; Merck KGaA). Mission TurboGFP Control transduction particles (Sigma-Aldrich; Merck KGaA) was used as a control. Lentiviral particles were transduced into HCC cells according to the manufacturer's instructions. The expression levels of GSTA2 mRNA and protein was examined by RT-PCR) and Western blot respectively.
The lentiviral open reading frame (ORF) clone of monomeric green uorescent protein (GFP)-tagged human GSTA2 (cat. no. RC202479L2) and lentiviral ORF control clone were purchased from OriGene Technologies, Inc. Lentiviral particles were produced by transfection into HEK293 cells using Lenti-vpak Lentiviral Packaging kit (OriGene Technologies, Inc). Suppression of GSTA2 was conducted by GSTA2-speci c Mission® short hairpin RNA (shRNA) lentiviral transduction particles (Sigma-Aldrich; Merck KGaA). Mission TurboGFP Control transduction particles (Sigma-Aldrich; Merck KGaA) was used as a control. Lentiviral particles were transduced into HCC cells according to the manufacturer's instructions. The expression levels of GSTA2 mRNA and protein was examined by RT-PCR) and Western blot respectively.
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