For overexpressing FOXM1, 50% confluent U251 and U87 cells in 6-well plates were cultured in 1 mL medium with lentivirus particles and 5 μg/mL polybrene (GeneChem). 12 h later, the cell medium was replaced with 2 mL fresh normal medium and cultured for another 48 h. Medium with 2 μg/mL puromycin was used for selecting stably transfected cells, and this puromycin-containing medium was refreshed every 3 days for at least a 9-day selection process. For FOXM1 transient knockdown, 50% confluent U251, U87, and LN229 cells were transfected with FOXM1-short interfering RNA (siRNA) oligonucleotide (CUCUUCUCCCUCAGAU AUAdTdT) or control siRNA (RiboBio, Guangzhou, Guangdong, China). After transfection for 12 h, fresh normal medium was added, and cells were cultured for another 48 h before performing the following experiments.
Polybrene
Manufactured by Genechem
Sourced in China, United States
Polybrene is a cationic polymer commonly used as a transfection reagent in cell culture applications. It facilitates the uptake of nucleic acids, such as DNA or RNA, into cells by neutralizing the negative charges on the cell surface and the nucleic acids, thereby enhancing the efficiency of transfection.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced infection solution, by Genechem (19 mentions)
Puromycin, by Thermo Fisher Scientific (13 mentions)
Puromycin, by Merck Group (12 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (12 mentions)
Lentivirus, by Genechem (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Opti mem, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (8 mentions)
Puromycin, by Genechem (5 mentions)
Lv nc, by Genechem (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (4 mentions)
Lentiviral vector, by Genechem (4 mentions)
Shrna mttfa, by Genechem (3 mentions)
Control shrna, by Genechem (3 mentions)
Te2000 u, by Nikon (3 mentions)
293t cell, by American Type Culture Collection (3 mentions)
Hiperfect transfection reagent, by Qiagen (3 mentions)
Lipo3000, by Thermo Fisher Scientific (3 mentions)
Control sirna, by GenePharma (3 mentions)
176 protocols using polybrene
1
FOXM1 Overexpression and Knockdown Protocols
2
Overexpression of Sirt3 in MSCs
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The cells were transduced with lentiviral particles encoding rat Sirt3 or control vector as previously described (Pi et al., 2019 (link)). Briefly, cells were plated at 1.5 × 105/well in six-well plate and incubated at 37°C for 18 h, and then, cells were transduced with lentivirus-expressing Sirt3 in the presence of 4 μg/ml polybrene (Genechem Co. Ltd., China) for 12 h. Sirt3 over-expressed MSCs were used in subsequent experiments.
3
Lentiviral Knockdown of VEGF-C in MSCs
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The lentivirus particles for knockdown of VEGF‐C were purchased from GenePharma. MSCs were infected with the described lentiviral vectors supplemented with 10 mg mL−1 polybrene (GeneChem). After transfection, MSCs were selected in medium containing 2 mg mL−1 puromycin.
4
Lentiviral Overexpression of Linc00961
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Lentivirus vector overexpressing Linc00961 (Lv-Linc00961) was designed and constructed by Shanghai GeneChem Co., Ltd. Briefly, the full length of Linc00961 was chemically synthesized and cloned into the BamHI and AgeI sites of a CV146 lentivirus core vector. Subsequently, CV146-Linc00961 (20 µg) and lentiviral packaging helper plasmids, Helper 1.0 (15 µg) and Helper 2.0 (10 µg; both Shanghai GeneChem Co., Ltd.), were co-transfected into 293T cells using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). The cell supernatant was collected 48 h later, and subjected to centrifugation (4°C, 4,000 x g, 10 min) in order to concentrate and purify Lv-Linc00961. Cells stably expressing Linc00961 were constructed by transfecting 5×105 TU Lv-Linc00961 or Lv-control (containing empty CV146 vector) into 2×106 A375 or SK-MEL-28 cells cultured in 1 ml enhanced infection solution (Shanghai GeneChem Co., Ltd.). Then, 5 µg polybrene (Shanghai GeneChem Co., Ltd.) was added in each well according to the manufacturer's protocol (17 (link)). Transfected cells were harvested 72 h later, and subsequently treated with puromycin (2 µg/ml) in order to isolate the Linc00961-stably-expressing cells.
5
Hypoxia-Induced Angiogenesis and Metastasis
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Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin were purchased from Hyclone (Logan, USA). Foetal bovine serum (FBS) was purchased from Gibco (South America). Anti-HIF1α monoclonal antibodies were purchased from CST (Boston, USA). Anti-VEGF monoclonal antibodies were purchased from RD (Minneapolis, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, New Super ECL Assay kit, and anti-β-actin antibodies were purchased from KeyGEN BioTECH (Nanjing, China). Transwell chambers and Matrigel were purchased from Corning Life Sciences (8-μm pores, Tewksbury, MA, USA) and BD Biosciences (San Jose, CA, USA), respectively. The QRT-PCR kit was purchased from Takara (Shiga, Japan). The Cell Counting kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. The BCA Protein Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). Recombinant lentiviral expression vectors (LV-HIF1α, LV-Con, LV-ShHIF1α, and LV-Scramble), and polybrene were obtained from Genechem (Shanghai, China). BALB/c nude mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd (Shanghai, China).
6
Lentiviral Transduction of Cancer Cells
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Pre-miR-506 lentivirus, miR-506 inhibitor lentivirus, UHRF1-overexpressing lentivirus, sh-UHRF1 lentivirus, KISS1-overexpressing lentivirus and PI3K-overexpressing lentivirus were purchased from GeneChem (Shanghai, China). Infection was performed using polybrene (GeneChem). A lentivirus infection efficiency of more than 80% was considered successful. According to the manufacturer’s instructions, the multiplicity of infection for HCT116 cells was 10, and the multiplicity of infection for SW480 cells was 30. After 48 h of infection, the cells were digested for further cell culture. All experiments were performed according to the manufacturer’s instructions.
7
Transfection of KDM6B and RNA interference in Gastric Cancer cells
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Roche transfection reagent (2 μl per sample; Roche, Basel, Switzerland) was used to transfect KDM6B or KDM6B (H1390A) (Addgene, Watertown, MA, USA), as recommended. Cells were collected 48 h post-transfection for further analysis. Lipofectamine 2000 (5 μl per sample; Invitrogen, Carlsbad, CA, USA) was used to transfect siRNA (5 μl per sample) into GC cells (4 × 105 cells per sample) as recommended. Cells were collected 72 h post-transfection. GC cells were inoculated into 24-well plates and 24 h later, the virus (containing shKDM6B or shNC; Genechem, Shanghai, PR China) was transfected into GC cells with a complex of infection (MOI) of 100. Polybrene (Genechem) was used to increase efficiency of transfection. The medium was changed 24 h after transfection and puromycin (2 μg/ml; Gibco) was added. Three days later, transfection efficiency was calculated using RT-PCR and protein blotting. The sequences of siRNAs and shRNAs used in the experiments are listed in Supplementary Material Table S1 . The final concentration of KDM6B inhibitor GSK-J4 (HY-15648B, MCE, Monmouth Junction, NJ, USA) used in this study is 1 μM, and CXCR4 inhibitor AMD3100 (HY-10046, MCE) is 100 nM.
8
Lentiviral Infection and Knockdown of lncRNA 604
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First, we used LV-lncRNA 604-ctrl and LV-lncRNA 604-RNAi-ctrl to infect HCT116 and HT29 cells. Because the lentiviral carried luciferin, we used fluorescein potassium salt to excite the fluorescence, and then observe the infection efficiency through the microscope. HCT116 and HT29 cells were infected with lentivirus (LV)-lncRNA 604, LV-lncRNA 604-RNA interference (RNAi) at a multiplicity of infection (MOI) of 20 and 10 µg/ml polybrene (Shanghai GeneChem Co, Ltd.).
After lentiviral infection for 8 h, these cells were maintained with normal RPMI-1640 medium for 24 h. Subsequently, these cells were incubated in RPMI-1640 with 2 μg/ml puromycin (Gibco-BRL, Gaithersburgh, MD, USA). The knockdown and overexpression efficiency of lncRNA 604 was further analyzed by RT-PCR.
After lentiviral infection for 8 h, these cells were maintained with normal RPMI-1640 medium for 24 h. Subsequently, these cells were incubated in RPMI-1640 with 2 μg/ml puromycin (Gibco-BRL, Gaithersburgh, MD, USA). The knockdown and overexpression efficiency of lncRNA 604 was further analyzed by RT-PCR.
9
Lentiviral Modulation of miR-25-3p and BTG2 in Breast Cancer
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The miR-25-3p mimics and inhibitor lentivirus were constructed by Genechem (Shanghai, China) to overexpress or knockdown miR-25-3p in breast cancer cells. The GV272 empty construct (miR-NC) served as a negative control. Target cells (2 × 105) were infected with 1 × 106 lentivirus transducing units in the presence of 10μg/ml polybrene (Genechem, Shanghai, China). The lentiviral vector containing BTG2 DNA sequence and siRNA for BTG2 were constructed by Genepharma (Shanghai, China). When MDA-MB-231 and Sum-1315 cells grew to 40–50% confluence, cells were infected with lentiviral vectors miR-NC, miR-25-3p mimics and inhibitor respectively. The lentiviral vectors were transfected into BC cells with the multiplicity of infection (MOI) of 20 to the MDA-MB-231 and Sum-1315. Stable cell lines were selected by using 3μg/ ml puromycin (Sigma, USA) for 1 week.
10
Lentiviral Modulation of FKBP51 in ESCs
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The ESCs were transfected with cDNA-FKBP51 (GV230 vector) and shRNA-FKBP51 (GV248 vector) packaged into a lentivirus purchased from GeneChem BioTECH (Shanghai, China) to overexpress and knockdown FKBP51 expression. The cDNA sequences were commercially designed, and the sequences for the shRNA against FKBP51 have been described previously (Pei et al. 2009) . After optimizing the experimental conditions, ESCs were seeded onto 24-well plates (30,000 cells/well) and were transfected using 5 μg/mL polybrene (GeneChem) with cDNA-FKBP51 or shRNA-FKBP51 using empty vectors as the respective controls (MOI: 50). The transfection efficiency was examined by flow cytometry. The overexpression and suppression rates were determined by performing Western blotting. FKBP51 cDNA was cloned into pcDNA3.1(+). In the rescue experiments, ESCs, grown to 60% confluence, were transfected using Lipofectamine 2000 (Invitrogen) with pcDNA3.1(+)/FKBP51 (3 μg in a 6-well plate) and empty pcDNA3.1(+). All transfections were performed in triplicate and repeated three times.
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