Two siRNAs against human SRC (GenBank accession no. NM_005417) were designed as described previously (Reynolds et al.,2004 ; Ui‐Tei et al.,2004 ). The 19 nucleotide target sequences (SRC‐siRNA1: position 1489–1507 and SRC‐siRNA2: position 1684–1702) were synthesized into 64–65 mer oligonucleotides with BamHI/HindIII overhangs (Sigma Aldrich) and cloned into the expression vector pSilencer 3.0‐H1, containing pmaxGFP (Ambion Inc.). All clones were purified using an EndoFree Plasmid Maxi Kit (Qiagen Ltd) and sequenced (Geneservice Ltd). hASMC were transfected using the Basic Nucleofector® Kit and nucleofector device (Amaxa Biosystems). After 72 h, the transfection efficiency was >90%, confirmed by fluorescence microscopy.
Basic nucleofector kit
Manufactured by Lonza
Sourced in Switzerland
The Basic Nucleofector Kit is a laboratory equipment designed for the transfection of cells. It enables the introduction of nucleic acids, such as plasmids or small interfering RNAs, into a variety of cell types through electroporation. The core function of the kit is to facilitate efficient gene transfer and expression without compromising cell viability.
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Lab products found in correlation
Pcmv3, by Sino Biological (2 mentions)
Pcmv3 ocln plasmid, by Sino Biological (2 mentions)
27mer oas1 sirnas, by OriGene (2 mentions)
27mer oas2 sirnas, by OriGene (2 mentions)
27mer oas3 sirnas, by OriGene (2 mentions)
27mer oasl sirnas, by OriGene (2 mentions)
27mer rnasel sirnas, by OriGene (2 mentions)
27mer occludin sirnas, by OriGene (2 mentions)
27mer tjp1 sirnas, by OriGene (2 mentions)
Amaxa nucleofector technology, by Lonza (2 mentions)
Anti occludin 27 mer sirna h, by OriGene (2 mentions)
Anti caveolin 1 27 mer sirna h, by OriGene (2 mentions)
Anti alix sirna h, by Santa Cruz Biotechnology (2 mentions)
Nucleofector 2, by Lonza (2 mentions)
Nucleofector device, by Lonza (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Effectene kit, by Qiagen (1 mentions)
Cell line nucleofector kit, by Lonza (1 mentions)
Amaxa nucleofection, by Lonza (1 mentions)
11 protocols using basic nucleofector kit
1
Silencing Human SRC Gene Expression
2
Electroporation of Primary Hippocampal Neurons
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Primary hippocampal neurons were electroporated in suspension immediately after dissociation, using the Basic Nucleofector Kit for primary neurons on the O-05 program (Amaxa Biosystems, Cologne, Germany). Neurons were electroporated with the constructs of interest (800 ng pmRFP-N1 human cofilin wt, 400 ng pmRFP-N1 human cofilin S3A, 400 ng pmRFP-N1 human cofilin S3E/1,000,000 neurons) at the 4D-NucleofectorTM Core Unit (Amaxa), and plated on glass coverslips, or on plastic Petri dishes, as described above. Electroporated neurons were used at 3 DIV.
3
Pericyte Transfection and Gene Silencing
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Pericytes were transfected using Amaxa Nucleofector Technology and the Basic Nucleofector Kit (Lonza, Switzerland, EU, Cat# VPI-1001) as described [16 (link)]. For ocln overexpression, cells were transfected with 1 μg PCMV3-OCLN plasmid (Sino Biological, Wayne, PA, Cat# HG15134-UT) or with PCMV3 (Sino Biological, Cat# CV011) as a negative control. For gene silencing, cells were transfected with 0.5 μg per 106 cells of the following small interfering RNA (siRNA): 3 unique 27mer OAS1-siRNAs (OriGene, Rockville, MD, USA, Cat# SR303266), 3 unique 27mer OAS2-siRNAs (OriGene, Cat# SR303267), 3 unique 27mer OAS3-siRNAs (OriGene, Cat# SR303268), 3 unique 27mer OASL-siRNAs (OriGene, Cat# SR305676), 3 unique 27mer RNaseL-siRNAs (OriGene, Cat# SR304081), 3 unique 27mer occludin-siRNAs (OriGene, Cat# SR303274), 3 unique 27mer TJP1-siRNAs (OriGene, Cat# SR322042), or trilencer-27 universal scrambled (SCR) silencer siRNA duplex (OriGene, Cat# SR30004) as a negative control.
4
CRISPR-Mediated Knockdown of IL-8 and Cxcl15
5
Silencing of Cav-1, Occludin, and Alix
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Cav‐1, ocln, and Alix silencing was performance using the following small interfering RNA (siRNA): anti‐occludin 27‐mer siRNA (h): Cat# SR321170, anti‐caveolin‐1 27‐mer siRNA (h): Cat# SR319567 (OriGene, Rockville, MD, USA), and anti‐Alix siRNA (h): Cat# sc‐60149 (Santa Cruz Biotechnology, Dallas, TX, USA). Trilencer‐27 universal scrambled (SCR) silencer negative control siRNA Cat# SR30004 (OriGene) was used as nonspecific control siRNA. Pericytes were transfected by Amaxa Nucleofector Technology with 1 µg siRNA or control siRNA per 106 cells using the Basic Nucleofector Kit originally designed for primary mammalian endothelial cells (Lonza, Switzerland, EU, Cat# VPI‐1001). Next day, cells were washed and allowed to recover in normal pericyte medium. For ocln overexpression experiments, ocln cDNA cloned into the pCMV6 plasmid was obtained from OriGene (Cat# RC206468). As a negative control, the pCMV6 plasmid (OriGene Cat# PS100001) was used. Cells were transfected with 2 µg of ocln overexpressing vector per 106 cells or negative control vector by using Amaxa Nucleofector Technology.
6
Overexpressing HES5 in SMC-EC Differentiation
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Plasmid pCMV6-HES5 (RG215311, Origene) containing full length human HES5 cDNA fragment was used to overexpress HES5 at day 2 of differentiation from PR-SMCs to SMC-ECs. Empty pCMV6 vector was used as negative control. Cells were transfected with pCMV6-HES5 or pCMV6 using Basic Nucleofector Kit (LONZA) as specified by manufacturer. The expression of the endothelial markers was evaluated at the transcriptional and protein levels after 72 hours of nucleofection.
7
Transfection and Gene Silencing in Pericytes
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Pericytes were transfected using Amaxa Nucleofector Technology and the Basic Nucleofector Kit (Lonza, Switzerland, EU, Cat# VPI-1001) as described [15 (link)]. For ocln overexpression, cells were transfected with 1 μg PCMV3-OCLN plasmid (Sino Biological, Wayne, PA, Cat# HG15134-UT) or with PCMV3 (Sino Biological, Cat# CV011) as a negative control. For gene silencing, cells were transfected with 0.5 μg per 106 cells of the following small interfering RNA (siRNA): 3 unique 27mer OAS1-siRNAs (OriGene, Rockville, MD, USA, Cat# SR303266), 3 unique 27mer OAS2-siRNAs (OriGene, Cat# SR303267), 3 unique 27mer OAS3-siRNAs (OriGene, Cat# SR303268), 3 unique 27mer OASL-siRNAs (OriGene, Cat# SR305676), 3 unique 27mer RNaseL-siRNAs (OriGene, Cat# SR304081), 3 unique 27mer occludin-siRNAs (OriGene, Cat# SR303274), 3 unique 27mer TJP1-siRNAs (OriGene, Cat# SR322042), or trilencer-27 universal scrambled (SCR) silencer siRNA duplex (OriGene, Cat# SR30004) as a negative control.
8
Reconstitution of Viruses from BACs
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Viruses were reconstituted from BACs by transfection of 0.5 × 106 epithelial cells, 2 × 106 HFFF cells, or 2 × 106 HFFF-Tet cells. HFFF and HFFF-Tet cells were transfected by electroporation, using program T16 of the Nucleofector II (Amaxa) and a basic Nucleofector kit (Lonza), according the manufacturer's instructions. On occasions when transfected HFFF or HFFF-Tet cultures were <70% confluent after overnight recovery, additional nontransfected cells were added. RPE-1 cells were transfected by chemical transfection using an Effectene kit (Qiagen), according to the manufacturer's instructions for transfection of adherent cells. The efficiencies of transfection of fibroblast and epithelial cells differed only marginally, with 20 to 35 plaques formed per electroporation in HFFF or HFFF-Tet cells and 50 to 60 plaques formed in RPE-1 cells.
9
Neoantigen Expression in HCMV Vectors
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As a neoantigen for expression in TB40/E-BAC derived vectors, we used human papillomavirus type 16 (HPV-16) consensus E6/E7 fusion protein (ConE6E7, GenBank accession number: FJ229356) (46 (link)). In addition, the HLA-A2-binding peptide E629−38 (TIHDIILECV ) derived from the E6 protein of HPV-16 (47 (link)) was fused with an AA-linker (AATIHDIILECV ) to the C-terminus of HCMV IE1 (E6peptideIE1) or HCMV UL83 (E6peptideUL83). The corresponding sequences were synthesized and verified by Integrated DNA Technologies (IDT). The synthesized E6/E7 encoding sequence was digested with EcoRI and Kpn-I and cloned into the expression vectors pEF6/V5-His A and pcDNA™3.1 (+). These constructs were named pEF6E6/E7EcoRI and pcDNAE6/E7Kpn-I, respectively. Recombinant HCMV was generated using BAC technology as previously described (48 (link)). All recombinant BAC clones were confirmed by PCR and DNA-sequencing of the target area. Viruses were reconstituted from BACs by electroporation of 1 × 106 Fi301 cells using program A24 of the Nucleofector II (Amaxa) and a basic Nucleofector kit (Lonza), according the manufacturer's instructions.
10
Silencing and Overexpressing Junctional Proteins
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