Mammalian expression vectors with different oncogene inserts and control vectors were transfected into Phoenix cells cultured in DMEM media, and Lipofectamine was used as the transfection agent (Invitrogen, NY, USA) along with viral packaging vectors. The viral supernatant was harvested after 48 hours and used to infect the growing cultures of HMEC 5.6 RAB25- and HMEC 2.6 RAB25- cells by using a polybrene reagent (Millipore, MA, USA). The resulting viral supernatant after 48 hours was collected and introduced to a growing culture of HMEC 5.6 RAB25- cells with the aid of polybrene reagent (Millipore, MA, USA). The transfected cells were subjected to Hygromycin selection (20 μg/ml) in vitro (Gold Biotechnology, MO, USA) over the next few weeks. The surviving clones were harvested separately by using sterile Cloning Cylinders (Fisher Scientific, MA, USA) and seeded in separate tissue culture plates.
Polybrene reagent
Manufactured by Merck Group
Sourced in United States, Germany, China
Polybrene reagent is a cationic polymer commonly used in cell and molecular biology applications to facilitate the transfection of nucleic acids, such as DNA or RNA, into cells. Its primary function is to enhance the efficiency of nucleic acid uptake by cells during transfection experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g, by Addgene (6 mentions)
Pspax2, by Addgene (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Puromycin, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (2 mentions)
X tremegene 9 dna transfection reagent, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Pmdg2 g, by Addgene (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
Prsv rev, by Addgene (1 mentions)
Pcmvr8, by Addgene (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Recombinant murine il 3, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Effectene reagent, by Qiagen (1 mentions)
Y 27632 2hcl, by Selleck Chemicals (1 mentions)
Vsv g, by Addgene (1 mentions)
Expi293 expression media, by Thermo Fisher Scientific (1 mentions)
26 protocols using polybrene reagent
1
Oncogene Transduction and Selection of HMEC Cells
2
Transfection of Endometrial Stromal Cells
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Eutopic endometrial stromal cells were seeded in 6-well plates and grown to 60–70% confluence. Cells were then transfected with the control recombinant lentivirus vector (Lv-control), siS100A6 lentivirus vector (Lv-siS100A6), or with no vector (untreated control) using Polybrene® reagent (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in accordance with the manufacturer's protocol. All transfections were performed in triplicate. Transfected cells were cultured at 37°C in an atmosphere containing 5% CO2 for 4 days, and were subsequently used for a range of analytical experiments.
3
Targeting HOTAIR Enhances Cisplatin Sensitivity in NSCLC
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Six-week athymic BALB/c mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. The animal experiments were approved by The Institutional Review Board of the Affiliated Tumor Hospital of Xinjiang Medical University and were untaken in accordance with National Institutions of Health Guide for Care and Use of Laboratory Animals. H1299/DDP cells were stably transfected lentiviral particles encoding shRNA against HOTAIR (sh-HOTAIR; Neuron Biotech, Shanghai, China) or the negative control (sh-NC) using Polybrene reagent (Sigma). Equal numbers (106) of transfected H1299/DDP cells/0.2 mL were subcutaneously injected in subcutaneous area of flanks (5 mice per group) for 35 days. One week later after transplantation, xenograft mice were subjected to intra-peritoneal injection of DDP at a dose of 3.0 mg/kg body weight or phosphate buffer solution (PBS; pH 7.4) every 7 days from the 7th day. Xenograft experiments were divided into three groups: sh-NC+PBS, sh-HOTAIR+PBS, sh-NC+DDP, and sh-HOTAIR+DDP. The tumors were measured with a caliper once 7 days, and the mice were practiced with euthanasia on day 35. The tumor volume was calculated using the formula: 0.5 × l × w2 (l is the length of tumor and w is the width of tumor). And the weight of tumors was evaluated with electronic balance. Immediately, the tumors were frozen in −80°C for further isolation of total RNA.
4
Lentiviral Transduction of Cell Lines
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All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (FBS) at 37 °C with 5% CO2. Cell lines were obtained from ATCC (catalogue numbers A549 – CCL-185; H1299 – CRL-5803; 143B – CRL-8303; U2OS – HTB-96; MDA-MB-468 – HTB-132; RPE-1 – CRL-4000); A9 cells were a gift from the B. Manning laboratory. All cell lines regularly tested negative for mycoplasma. To generate cells with stable transgene expression, Lenti-X 293 T cells at 75% confluency were transfected using X-tremeGENE 9 DNA transfection reagent (Sigma). The lentiviral plasmids used were pRSV-Rev (Addgene #12253), pMDLg/pRRE (Addgene #12251) and pMDG2.G (Addgene #12259) from Didier Trono. For the YFP-DHFR reporter, the donor expression plasmid pLJM1-FLAG-YFP-DHFR30 (link) was used. For the mVenus-Gem1 reporter, the donor expression plasmid pLenti-puro-mVenus-Gem1 was used. After 48 h, lentivirus was collected by removing the culture medium from the Lenti-X 293 T cells and passing it through a 0.45 μm filter. The target cell lines at 50–60% confluency were then infected using 3 ml virus with polybrene reagent (Sigma). After 24 h, virus was removed and cells were allowed to recover in virus-free medium for 24 h. Selection was then initiated using puromycin at a concentration of 2 μg ml−1.
5
Lentiviral Transduction and Selection of EMSCs
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293T cells were transfected with lentiviral transfer vectors, pCMVR8.74 (Addgene #22036, a gift of Didier Trono), and pMD2.G (Addgene #12259, a gift of Didier Trono) at a ratio of 3:2:1 using the Lipofectamine 3000 transfection reagent (Thermo, L30000015). The supernatant containing lentiviral particles was collected 48 and 72 h after transfection. EMSCs were transduced with corresponding lentiviral particles supplemented with polybrene reagent (Sigma, TR1003, 10 μg/ml). 48 h later, cells were treated with the corresponding antibiotic puromycin (Sigma, P9620, 500 ng/ml) or G418 (Thermo, 10131027, 500 µg/ml) for two weeks to enrich transduced cells.
6
Investigating TP73-AS1 in Hepatocellular Carcinoma Xenograft
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HCC‐70 cells were infected by lentiviral particles encoding shRNA against TP73‐AS1 (sh‐TP73‐AS1; Neuron Biotech, Shanghai, China) or the negative control (sh‐NC) using Polybrene reagent (Sigma, St Louis, MO, USA). The six‐week‐old BALB/c nude mice were purchased from HFK Bio‐Technology (Beijing, China), and randomly divided into two groups (n = 6). Stably transfected HCC‐70 cells (2.5 × 106 cells) were then subcutaneously injected into the right flanks of mice. After inoculation, tumor volume was monitored every week with the formula: 0.5 × length × width.2 On the fifth week, the mice were euthanatized and the tumors surgically separated. The tumor weight was subsequently recorded and the tumors snap‐frozen in liquid nitrogen for further RNA or protein detection. All animal procedures were carried out in accordance with the Animal Care Committee of Changji Huizu People's Hospital of Xinjiang.
7
Lentiviral Transduction of Target Cells
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Lentiviruses were produced in HEK293T cells by co-transfection with the viral vector and packaging plasmids (pMD2.G: Addgene, 12259; psPAX2: Addgene, 12260). Two days after transfection, viral supernatant was harvested, filtered through a 0.45 μm filter, and added to target cells in the presence of polybrene reagent (Sigma, R-1003-G) at 4 μg/mL. The infected cells were selected with puromycin, hygromycin B, or blasticidin as indicated below.
8
Lentiviral Transduction and Inducible Expression
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For lentiviral transduction, HEK293T cells were transfected using Effectene reagent (QIAGEN), using pCMV-dR8.2 dvpr packaging vector and pMD2.G VSV-G envelope. Ba/F3 cells were transduced with lentivirus encoding pFTRE GFP (control construct), T SPECC1L–NTRK2, and FL SPECC1L–NTRK2 by spin infection and Polybrene reagent (Sigma-Aldrich). GFP-positive (fusion-expressing) Ba/F3 cells were sorted by flow cytometry following transduction. Cell lines were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 0.5 ng/mL recombinant murine IL-3 (Peprotech), at 37°C and 5% CO2. For induction, 1 µg/mL of doxycycline was added to cultures, and fusion-expressing cells were maintained in RPMI supplemented with 10% FBS following selection.
9
Lentiviral Transduction of hPSCs
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The cDNA of NUP98-KDM5A was obtained in a pMSCV vector (kindly provided by Dr David Allis; The Rockefeller University, New York, USA). The cDNA was sub-cloned using standard procedures into pRRL-EF1a-PGK-NEO vector (kindly provided by Prof. Trono, EPFL, Lausanne, Switzerland) for the expression in hPSCs. To generate the lentiviral vectors, HEK-293T cells were transfected with pRRL-EF1a-PGK-NEO (Control) or pRRL-EF1a-NUP98-KDM5A-PGK-NEO (NK5A) together with psPax2 (packaging vector) and VSV.G (envelope vector), (Addgene; Watertown, Massachusetts, USA) by standard calcium-phosphate transfection protocol. The supernatants were collected 48 h after transfection and used fresh supplemented with Polybrene Reagent (8 mg/mL, Sigma-Aldrich; St. Louis, Missouri, USA) and Y-27632 2HCl (10 μM, Selleckchem, Houston, TX, USA) to transduce hPSC (H9 and PBMC2-iPS4F8) in single cell suspension at the day of passage. After 2 days, the cells were selected with G418 (100 mg/mL; Invitrogen; Carlsbad, California, USA) for 5 days.
10
Establishing DLK1-Overexpressing Lentiviral System
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A lentivirus expressing green fluorescent protein (GFP) and harboring the DLK1 gene was constructed as the dlk1-overexpression (oe) group, while a lentivirus expressing GFP without the DLK1 gene was constructed as the control group (Shanghai GeneChem Co., Ltd., Shanghai, China). ALCs were seeded into six-well plates and grown to ~50% confluence prior to transfection. The ALCs were infected with 10 µl lentiviruses (1×108 U/ml) using polybrene reagent (Sigma-Aldrich; Merck KGaA) for 24 h, according to the manufacturer's protocol. Following transfection, 10 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) was added for 2 weeks to select the positively transfected cells. A monoclonal population of stably infected cells (termed dlk1-oe or control) were pooled and used for further experiments, respectively. The transfected cells were analyzed under a fluorescence microscope and confirmed by RT-qPCR and western blot analysis.
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