SVF was transduced with Mouse LentiORF Particles (pLenti-C-mGFP-P2A-Puro; Origene, Rockville, MD, USA): Mef2c (NM_025282, MR226865L4V), Gata4 (NM_008092, MR227022L4V), or Tbx5 (NM_011537, MR227369L4V) for overexpression, or LentiORF Control Particles (pLenti-C-mGFP-P2A-Puro, PS100093V, Origene) as an empty vector control. For silencing of mouse Mef2c, LentiORF lentiviral particles of mouse Mef2c-specific short hairpin RNA (TL511428V, Origene) or scrambled control were used. Cells at ~ 50% confluence on days 5 to 7 after seeding of SVF at 5000/cm2 on 12-well culture plates were incubated for 24 h in 500 µL of minimal medium at a multiplicity of infection of 10. Subsequently, the lentivirus-containing medium was replaced with fresh medium plus puromycin (5 µg/mL, A11138-03, Gibco) to select for successfully transduced cells. puromycin-resistant colonies were observed under a microscope.
Plenti c mgfp p2a puro
Manufactured by OriGene
Sourced in United States
PLenti-C-mGFP-P2A-Puro is a lentiviral vector that allows for expression of mGFP and puromycin resistance genes. It contains a CMV promoter to drive the expression of the reporter and selection genes.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Maxi kit, by Qiagen (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Bradford assay kit, by Bio-Rad (1 mentions)
Hrp conjugated rabbit anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hek293t cell line, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Low protein binding filter, by Merck Group (1 mentions)
Hexadimethrine bromide, by Merck Group (1 mentions)
Suresilencing shrna plasmid, by Qiagen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
9 protocols using plenti c mgfp p2a puro
1
Lentiviral Transduction of Adipose Stromal Vascular Fraction
2
Genetic Manipulation of Fibroblast Cell Lines
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Mouse NIH 3T3 fibroblasts were stably transfected with Lipofectamine 2000 (Invitrogen) with mouse Sostdc1 (NM_025312) SureSilencing shRNA Plasmids (Qiagen), or scrambled sequence negative control plasmids (Qiagen) according to manufacturer's instructions. Cells were selected in fresh complete media containing G 418 (400 μg/ml; Sigma-Aldrich) and were maintained in G 418 (200 μg/ml). Sostdc1 protein levels in conditioned media were assessed following PNGase F (New England Biolabs) treatment for 1 h to remove glycosylations.
Linear morphoea normal primary dermal fibroblasts were stably transduced with: ADAMTS8 (NM_007037) Human LentiORF Particles (pLenti-C-mGFP-P2A-Puro; OriGene) for overexpression, or LentiORF Control Particles (pLenti-C-mGFP-P2A-Puro; OriGene) as an empty vector control. Cells at ~50% confluency were incubated overnight in complete media with polybrene (8 μg/ml; Sigma-Aldrich) at an MOI of 6. Subsequently, the lentivirus-containing media was replaced with fresh complete media for 24 h. Cells were then selected in fresh complete media plus puromycin (2 μg/ml; Sigma-Aldrich) according to a puromycin titration (data not shown) to select for successfully transduced cells. After ~10 days, with selection media being replaced routinely every 3-4 days, puromycin-resistant colonies were expanded and used in experimentation.
Linear morphoea normal primary dermal fibroblasts were stably transduced with: ADAMTS8 (NM_007037) Human LentiORF Particles (pLenti-C-mGFP-P2A-Puro; OriGene) for overexpression, or LentiORF Control Particles (pLenti-C-mGFP-P2A-Puro; OriGene) as an empty vector control. Cells at ~50% confluency were incubated overnight in complete media with polybrene (8 μg/ml; Sigma-Aldrich) at an MOI of 6. Subsequently, the lentivirus-containing media was replaced with fresh complete media for 24 h. Cells were then selected in fresh complete media plus puromycin (2 μg/ml; Sigma-Aldrich) according to a puromycin titration (data not shown) to select for successfully transduced cells. After ~10 days, with selection media being replaced routinely every 3-4 days, puromycin-resistant colonies were expanded and used in experimentation.
3
Site-Directed Mutagenesis of OAT Transporters
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We used plasmid pLenti-C-mGFP-P2A-Puro from Origene with SLC22A6 (OAT1) or SLC22A8 (OAT3) gene. To prepare allelic variants of these genes, we used Geneart site-directed mutagenesis kits. We designed the mutagenesis primers for each variant and performed mutagenesis PCR with template plasmid methylation. The primer used for site-directed mutagenesis is shown in Table 1 . The reaction mixture included 4.0% DMSO and 0.3 mM MgSO4. After verifying the PCR product, a recombination reaction was carried out and further transformed the dH5alfa bacteria with a prepared plasmid for each variant. After plasmid amplification in bacteria, we isolate the plasmid DNA using Qiagen Maxi Kits. We verified the results by sequencing the gene’s coding sequence (Sequenation laboratory, Faculty of Science, Charles University, Prague, CZ), and this one was compared with the sequence published in the Ensembl database [34 (link)].
4
Lentiviral Transduction of CPSF1
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The pLenti-C-CPSF1-mGFP-P2A-Puro and pLenti-C-mGFP-P2A-Puro empty vectors were purchased from OriGene (OriGene Technologies, Inc., Rockville, MD, USA). Lentiviral particles were prepared for CPSF1 and empty vector expression using 293T cells as the packaging cells. SCC090 and SCC17B cells were infected with viral supernatants for 24 h at 37°C in the presence of 8 μg/ml Polybrene (hexadimethrine bromide; Sigma). The transfected cells were selected using 1 μg/ml Puromycin (Invivogen, San Diego, CA, USA).
5
Synuclein and Cofilin Expression Vectors
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pCMV6-Entry vectors encoding FLAG-tagged human α-synuclein (RC210606) and β-synuclein (RC215165), pCMV6-AC-GFP vectors encoding TurboGFP-tagged human α- (RG210606) and β-synuclein (RG215165), pLenti-C-mGFP-P2A-Puro lentiviral control particles (PS100093V) and pLenti-C-mGFP-P2A-Puro encoding mGFP-tagged human cofilin (RC203585L4V) were from Origene. The mito-Keima construct (mt/mKeima/pIND(SP1)) was a gift from Dr. A. Miyawaki (RIKEN Brain Science Institute, Japan). The Keima construct (mKeima-Red-N1) was a gift from Dr M. Davidson (Addgene, 54597). The pEGFP-C1 vector was a gift from Dr P. Vangheluwe (KU Leuven). Cloning of Keima and mito-Keima cDNA into lentivirus and production of lentiviral particles were described before17 (link).
6
Lentiviral Transduction of OVCAR-3 Cells
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OVCAR-3 cells stably expressing GFP were generated for functional studies. For the production of lentiviral particles, 293 T cells were co-transfected with 5 μg of lentiviral vectors pLenti-C-mGFP-P2A-Puro (Origene, PS100093), 5 μg of pMD2-VSV-G ENV, 2.5 μg of pRSV-Rev, 2.5 μg of pMDLg/pRRE by using the calcium phosphate method. After 48 hours, the supernatant containing lentiviral particles was recovered, ultracentrifuged at 19.800 rpm on an SW28 rotor for 2 hours, and resuspended in phosphate-buffered saline (PBS) (500 μl for 20 ml of supernatant). OVCAR-3 cells were infected with 80 μl of viral suspension in a medium supplemented with polybrene (4 μg/ml) for 8 hours. Two consecutive rounds of infections were performed to improve the efficiency.
7
SKNMM^MYCN Cells Lentiviral Transduction
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SKNMMMYCN cells were transduced by a lentiviral vector (pLenti-C-mGFP-P2A-Puro) from Origene expressing either CUX2 (Cat# RC222063L4V) or GFP control construct (Cat# PS100093V). GFP-positive cells were sorted after 48 h and maintained in a selective media containing 1.5-μg puromycin per ml.
8
Characterization of SLC22A6 and SLC22A8
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The HEK293T cell line was purchased from Sigma (Cat. No.: 12022001-1VL). Cultivation medium DMEM, FBS, and supplements were purchased from Sigma Aldrich. Radiolabeled 14C uric acid MC-1394 was purchased from Hartmann analytic GmbH, Braunschweig, Germany. Plasmids with cloned SLC22A6 WT (wild type) and SLC22A8 WT pLenti-C-mGFP-P2A-Puro and mGFP primary antibody OTI2FG were purchased from Origene, Rockville, MD, USA. Mutagenesis was accomplished using Geneart Site-directed mutagenesis kits from Qiagen (Cat. No.: A13282), Hilden, Germany. Beta-actinin primary antibody was purchased from Cell Signaling (clone 8H10D10), Danvers, MA, USA. Rabbit anti-mouse HRP conjugated secondary antibody was provided by Invitrogen, cat. No.: A90-117P. Kits for plasmid DNA isolation were purchased from Qiagen, Hilden, Germany. Bradford assay kits were purchased from Biorad, Hercules, CA, USA. Cultivation plastic and were provided by VWR, Radnor, PA, USA. PVDF blotting membranes were purchased from Merck Immobilon (Cat. No. IPVH 07850). Other common chemicals came from Penta chemicals, Praha, Czech Republic or Sigma Aldrich, St. Louis, MO, USA.
9
Generation of KiSS1-overexpressing HepG2 Cells
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Transfection of the KiSS1 gene into 293T cells was conducted according to the manufacturer’s manual (Origene, USA). Briefly, for transformation, 293T cells were stably packaged with lentviral vector pLenti-C-mGFP-P2A-Puro (Origene) containing KiSS1 DNA (Origene). The lentivirus-containing medium was collected from 293T packaging cell line, the medium was filtrated with 0.22 µm low protein binding filter (Millipore, Germany). At the half-confluent of HepG2 cells then, the lentivirus-containing medium transferred to HepG2 cells (50%-60% confluency) with 10 µg/ml hexadimethrine bromide (Sigma-Aldrich, USA). After infection for 18 h, the lentivirus-containing medium was replaced with fresh cell culture medium and further incubated for 48 h. Transfectants were selected with antibiotics free-medium containing 10 µg/ml puromycin (Sigma-Aldrich) for 48 h, and was repeated 3 times. In this study, the KiSS1-overexpressing HepG2 cell line gene was named HepG2-KiSS1 cells. For vector control group, mock transfection was performed without adding only KiSS1 cDNA clone.
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