A lentiviral open reading frame plasmid Lv-TK1 (EX-C0529-Lv105) containing human TK1 mRNA complete sequence [PubMed cDNA clone MGC number: 3644] and a control plasmid (EX-NEG-Lv105) were obtained from GeneCopoeia, USA. The sequences of cloned plasmids were confirmed by DNA sequencing using 5’-GCGGT AGGCG TGTAC GGT and 5’-ATTGT GGATG AATAC TGCC as the forward and reverse primers, respectively. Pseudo lentiviral particles for TK1 overexpression and the control were produced with Lv-TK1 or the control plasmid and Lenti-Pac HIV expression packaging kit (GeneCopoeia, USA) on 293T cells according to manufacturer’s protocol. Pseudovirus titer was estimated on Hep3B cells under the selection of puromycin (0.5 μg/mL) as 1.6 × 107 and 1.2 × 107 transducing units/mL for TK1 and the control, respectively. Transduction of HL-7702 and Bel-7402 cells was carried out at a cell density of 50,000 in a 24-well plate with 80 μL pseudovirus stock solution plus poloxamer F108 (100 μg/mL, 10 μL) and polybrene 100 (100 μg/mL, 10 μL). After 24 h, the transduction media were replaced with the normal DMEM growth media, and cells were grown in a 6-well plate for 7 days. Western blot analysis and cell viability MTT assay with compound treatment were then carried out similarly as described in siRNA study.
Ex neg lv105
Manufactured by GeneCopoeia
Sourced in United States
The EX-NEG-Lv105 is a lab equipment product offered by GeneCopoeia. It serves as a negative control for gene expression studies. The core function of this product is to provide a baseline for comparison in experiments involving gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lenti pac hiv expression packaging kit, by GeneCopoeia (2 mentions)
Bxpc 3 cells, by American Type Culture Collection (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Virapower mix, by GeneCopoeia (1 mentions)
Polybrene reagent, by Merck Group (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
293ta packaging cells, by GeneCopoeia (1 mentions)
8 protocols using ex neg lv105
1
Lentiviral Transduction for TK1 Overexpression
2
Silencing Dab2 Using miRNA Plasmids
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The miRNA plasmids for silencing Dab2 (pcDNA™6.2-GW/±EmGFP-miR-Dab2) were constructed by following the protocol from the kit (K4936-00, Invitrogen). Human DAB2 (EX-M0885-lv105, GeneCopoeia), mouse Dab2 short isoform (p67) (EX-Mm02067-lv105, GeneCopoeia), mouse Dab2 long isoform (p96) (EX-Mm24887-lv105, GeneCopoeia) expression plasmids, and control plasmid (EX-NEG-lv105, GeneCopoeia) were used. The Mef2c expression plasmid was constructed by cloning the Mef2c coding sequence (CDS) into the pCS2+ vector and fused with a HA tag. Primers used in the study are listed in Supplementary Table 2 .
3
Engineered Colo-26 and BxPC3 Cell Lines for Protein Secretion Studies
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Colo-26 cells were provided by Prof. Zeiser, Freiburg, Germany. BxPC3 cells were purchased from ATCC. Cells were cultured in Dulbecco's modified eagle medium (DMEM; PAN Biotech) supplemented with 10 % fetal calf serum (FCS; PAN), 1 % non-essential amino acids, 1 % MEM vitamins, 1 % penicillin/streptomycin, and 10 mM HEPES (pH 7.5) at 37 °C in humidified air containing 5 % CO2. Serum-free cell-conditioned medium (CCM) of human BxPC3 pancreatic ductal adenocarcinoma cells for western-blot analysis (see below) was generated as described previously for the ovarian cancer cell line OV-MZ-6 24 (link). Murine Colo-26 colon cancer cells were transduced for stable luciferase expression as described previously for 291PC Burkitt lymphoma cells 25 (link). These cells were further transduced with the vector pReceiver-Lv105, constitutively expressing untagged human PSG1 under control of the cytomegalovirus (CMV) promoter (Genecopoeia, EX-Z8117-Lv105). pReceiver-Lv105 lacking the PSG1 gene served as an empty vector control (Genecopoeia, EX-NEG-Lv105). Puromycin selection (20 μg/ml for 14 d) was used for selection of stable transfectants. Serum-free cell-conditioned medium (CCM) of human BxPC3 pancreatic ductal adenocarcinoma cells and Colo-26 cells for western-blot analysis (see below) was generated as described previously for the ovarian cancer cell line OV-MZ-6 24 (link).
4
Generating Stable Overexpressing Cell Lines
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To generate stable mir-150-overexpressing cells, A2780, SKOV3, and ES2 cells were transduced with a lentivirus-expressing mir-150 or a negative control (Genechem, Shanghai, China), and selected using 0.5 μg/mL puromycin. After 1 week, puromycin-resistant cell pools with green fluorescent protein signals were identified using laser scanning confocal microscopy, collected, and then verified by real-time RT-PCR. To generate stable FoxP3-overexpressing, IRS1-overexpressing, mir-150 and IRS1 co-expressing, or mir-150 and IGF1R/IRS1 co-expressing cells, the lentiviral particles were first produced in HEK293T cells transfected with p-FoxP3 (EX-T8364-Lv105, GeneCopoeia, USA), p-IRS1 (EX-G0328-Lv105, GeneCopoeia), p-IGF1R (EX-L0202-Lv152-IGF1R, GeneCopoeia), or control plasmid (EX-NEG-Lv105, EX-L0202-Lv152, GeneCopoeia) along with psPAX2 (Addgene, Watertown, MA, USA) and pMD2.G (Addgene) plasmids. OC cells were then infected with lentiviral particles and selected using 0.5 μg/mL puromycin or 200 μg/mL hygromycin. After 1 week, puromycin or hygromycin-resistant cell pools were collected and verified by western blotting.
5
Lentiviral Vector Transduction of GC Cells
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The miR-939 expression vector (HmiR0533-MR03), the control vector for miR-939 (CmiR0001-MR03), the coding sequences of SLC34A2 expression vector (EX-A3175-Lv105), and the control vector for SLC34A2 (EX-NEG-Lv105) were purchased from the GeneCopoeia Company (Guangzhou, China). Cells transfected with empty vector were used as controls. The vectors were packaged using the ViraPower Mix (Genecopoeia, Guangzhou, China) in 293FT cells. After culturing for 48 h, the lentiviral particles in the supernatant were harvested and filtered by centrifugation at 500 g for 10 min, and then transfected into the GC cells.
6
Stable CD24 Overexpression in UM-SCC-74B
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CD24 mammalian expression vector (EX-G0006-Lv105-10) and a control vector (EX-NEG-Lv105) were purchased from Genecopoeia (Rockville, MD, USA). CD24 mammalian expression vector and control vectors were transfected into a growing culture of 293T cells with the aid of packing vectors and Lipofectamine transfection reagent (Invitrogen, NY, USA). The resulting viral supernatant after 48 hours was collected and introduced to a growing culture of UM-SCC-74B with the aid of polybrene reagent (Millipore, MA, USA). Stable UM-SCC-74B CD24-UP (upregulation) clones was obtained by Puromycin selection (3μg/μl) in vitro.
7
Lentiviral Overexpression of GPX4 in NSCLC
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Lentiviral vectors including GPX4 (CS-M0369-Lv105) and Negative Control (EX-NEG-Lv105) were purchased from GeneCopoeia. For lentivirus production, HEK293T packaging cells were transfected with 10 μg lentiviral vectors using the calcium phosphate method. After 48 h of incubation, the viral supernatant was collected and filtered. NSCLC cells were incubated overnight with the viral supernatant and supplemented with 10 μg/ml polybrene. Puromycin at a dose of 2 μg/ml was used to select the cell line overexpressing GPX4 stably.
8
Generating Stable Cell Lines for Signaling Pathway Analysis
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All stable cell lines were generated using lentiviral transduction. Alpha-casein (EX-S0252-Lv105) and empty vector control (EX-NEG-Lv105) expression plasmids were purchased from GeneCopoeia. Lentiviral particles were produced using the Lenti-Pac HIV expression Packaging kit and 293Ta packaging cells both obtained from GeneCopoeia as per manufacturers' instructions, and viral supernatant was used to infect target cells. Pre-made Cignal™Lenti reporter constructs purchased from Qiagen and used to infect target cells to generate stable reporter cell lines as per the manufacturer's instructions. These reporter constructs consist of a luciferase reporter gene fused to the applicable transcriptional response element (TRE) for each signalling pathway (details Table 1), and are used to quantitatively measure changes in signalling pathway activation in response to different stimuli. Two days following infection, successfully transduced cells were selected for using 2 μg/ml Puromycin or 1 mg G418 Geneticin® for 14 days.
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