1 × 106 KERA‐308 or 4 × 105 HEK293A cells were seeded in 6‐well plates (Greiner‐BioOne) 24 h prior to transfection. For KERA‐308 cells, 2 µg of hK14‐LaNtα31‐T2A‐mCherry or LaNt‐α31‐pSec‐Tag and 2 µl Lipofectamine 2000 (Thermo Fisher Scientific) were used. For HEK293A cells, either 1 µg pCAG‐Cre:GFP and 2 µl Lipofectamine 2000, 2 µg of pUbC‐LoxP‐LaNtα31‐T2A‐tdTomato and 5 µl Lipofectamine 2000, or 2 µg of pUbC‐LoxP‐LaNtα31‐T2A‐tdTomato, 1 µg of pCAG‐Cre:GFP and 7 µl Lipofectamine 2000 (Thermo Fisher Scientific), were mixed with 2 ml of Gibco™ Opti‐MEM™ Reduced Serum Medium (Thermo Fisher Scientific) and incubated for 10 min at room temperature. The DNA‐lipofectamine complex was added to the wells, and the media was replaced with DMEM high glucose after 6 h.
Lipofectamine 2000
Manufactured by Thermo Fisher Scientific
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3647 mentions)
Opti mem, by Thermo Fisher Scientific (3196 mentions)
Dual luciferase reporter assay system, by Promega (2953 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2201 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1504 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1226 mentions)
Penicillin, by Thermo Fisher Scientific (834 mentions)
Streptomycin, by Thermo Fisher Scientific (818 mentions)
Polybrene, by Merck Group (750 mentions)
Puromycin, by Merck Group (628 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (627 mentions)
Opti mem medium, by Thermo Fisher Scientific (542 mentions)
Dual luciferase reporter assay kit, by Promega (534 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (507 mentions)
Glutamax, by Thermo Fisher Scientific (471 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (440 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (400 mentions)
Puromycin, by Thermo Fisher Scientific (398 mentions)
Dual glo luciferase assay system, by Promega (391 mentions)
Rpmi 1640, by Thermo Fisher Scientific (383 mentions)
48 538 protocols using lipofectamine 2000
1
Transfection of KERA-308 and HEK293A Cells
2
Lentiviral and Retroviral Transduction Protocol
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Third‐generation lentivirus was generated by transient transfection of HEK293T cells with lentiviral plasmids (dCas9‐KRAB, pFgH1tUT), pMDL (packaging), RSV‐REV (packaging) and VSVg (envelope) plasmids complexed into liposomes using Lipofectamine 2000 (Thermo Scientific) diluted in OptimMEM (Thermo Scientific). Second‐generation lentivirus was generated via transient transfection of HEK293T cells with lentiviral pLVX, pPAX2 (packaging) and pMD2.G (envelope) plasmids complexed into liposomes using Lipofectamine 2000 (Thermo Scientific) diluted in OptimMEM (Thermo Scientific). Retrovirus was generated by transient transfection of HEK293T cells with pRetroX, gag‐pol (packaging) and VSVg (envelope) plasmids complexed into liposomes using Lipofectamine 2000 (Thermo Scientific) diluted in OptimMEM (Thermo Scientific). Lentiviruses or retroviruses were harvested 48 h later, filtered through a 0.45 μm filter and used to infect iBMDMs target cell lines. Cells were subsequently enriched for lentiviral plasmid expression via antibiotic selection (i.e., 5 μg/ml blasticIdin, 2.5 μg/ml puromycin) or cell sorting using a BD Biosciences Influx™ sorter.
3
Production of Reprogramming Viruses
4
Viral Interference Assays for Virus Replication
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Interference assays were performed using three different methods. (i) 293 cells were seeded at 1 × 10∧5 cells per well in 24-well plates and left to adhere overnight. Cells were transfected with 250 ng of R2_pcDNA/DEST40 vectors/well using 2 µl lipofectamine 2000 (ThermoFisher Scientific). 24 hpt cells were infected with E7 WT virus at MOI 0.01 for 48 hr. Virus-containing supernatants were titrated by EPDA on RD cells. (ii) RD cells were seeded at 1.5 × 10∧4 cells / well in 96-well plates and left to adhere overnight. Cells were co-transfected with 6.3 ng ncS variant RNA and 50 ng E7 luc replicon with ncR1_WT per well using 0.4 µl lipofectamine 2000 (ThermoFisher Scientific). Six hpt luciferase activity was quantified as described above. (iii) RD cells were seeded at 1.5 × 10∧4 cells/well in 96-well plates and left to adhere overnight. Cells were co-transfected with 42 ng EGFP expressing E7 replicon with the indicated R2 and ncR1 variants and 50 ng of luciferase expressing E7 with ncR1 of WT composition using 0.4 µl lipofectamine 2000 (ThermoFisher Scientific). Six hpt luciferase activity was quantified as described above.
5
Modulating miRNA and p21 Expression
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To silence a miRNA, cells (1 × 105 per mL) were transfected with 50 nM of anti-miRNA oligonucleotide (AMO) (AMO-miR-17-5p, 5′-CUACCUGCACUGUAAGCACUUUG-3′) or AMO-miR-17-5p-scrambled (5′-CAGUACUUUUGUGUAGUACAA-3′) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The AMO-miRNA was synthesized by GenePharma Co., Ltd., Shanghai, China). At 48 h after transfection, the cells were collected for later use.
To overexpress a miRNA, cells (1 × 105 per mL) were cultured in a six-well plate and transfected with 50 nM of the synthesized miRNA (GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). A total of 48 h after transfection, the cells were collected for later use.
To overexpress p21, p21 was cloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) using the sequence-specific primers (5′-CGGGATCCTGCCGAAGTCAGTTCCTTGT-3′ and 5′-C GTCTAGAGCACCTGCTGTATATTCAGC-3′). Then, GCSCs (1 × 105 per mL) were transfected with the recombinant pcDNA3.1 expressing p21 or plasmid alone using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. At different times after transfection, the cells were collected for later use.
To overexpress a miRNA, cells (1 × 105 per mL) were cultured in a six-well plate and transfected with 50 nM of the synthesized miRNA (GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). A total of 48 h after transfection, the cells were collected for later use.
To overexpress p21, p21 was cloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) using the sequence-specific primers (5′-CGGGATCCTGCCGAAGTCAGTTCCTTGT-3′ and 5′-C GTCTAGAGCACCTGCTGTATATTCAGC-3′). Then, GCSCs (1 × 105 per mL) were transfected with the recombinant pcDNA3.1 expressing p21 or plasmid alone using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. At different times after transfection, the cells were collected for later use.
6
Plasmid Transfection in Cultured Cells
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Cultured cells were transfected with polyethylenimine (Sigma-Aldrich, Switzerland) at a ratio of 3:1 PEI:DNA, or lipofectamine2000 (Thermofisher Scientific, Switzerland) following the manufacturer’s procedures at a ratio of 3:1 lipofectamine 2000:DNA. Briefly, 3 μL lipofectamine2000 was diluted in 100 μL Opti-MEM medium (Thermofisher Scientific, Switzerland) and incubated for 5 min at room temperature. The diluted lipofectamine2000 was then added to 1 μg of plasmid DNA diluted in 100 μL Opti-MEM medium. The mixture was incubated for 5 min at room temperature and then added directly to the cells at a confluency of about 90%. One day post-transfection the medium was replaced with Dulbecco’s modified eagle medium-high glucose (DMEM) containing 10% FBS and 2 mM L-glutamine. For transfections of eRF1, the cells were transfected 2 days before transfection with the reporter vector.
7
Nascent Protein Translation and RAN Translation Analysis
8
IFITM3 Regulation of Viral Env Expression
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Wild-type or IFITM3 KO HeLa cells (43 (link)) were seeded in 12-well plates at 300,000 cells per well and transfected with pCD-Env (1.2 or 0.5 μg) or pCMV-Xenogp85 (1.2 or 0.5 μg) using Lipofectamine 2000 (Thermo Fisher). Cotransfection of pCD-Env plasmid (1.4 μg) and negative-control siRNA (Ambion, Silencer Select, Negative Control no. 1) or siRNA against IFITM3 (Ambion, Silencer Select, s195035) using Lipofectamine 2000 was performed in HeLa cells seeded in 12-well plates at 300,000 cells per well. Cells were collected at 48 h posttransfection, lysed, and subjected to SDS-PAGE as described above. MEFs and IfitmDel MEFs were seeded in Lab-Tek II chamber slides (Thermo Fisher) at 15,000 cells per chamber and transfected with pCD-Env-EGFP (0.1 μg), with or without pQCXIP-FLAG-mIfitm3 (0.02 μg), using Lipofectamine 2000. At 48 h posttransfection, living cells were imaged using a TCS SP8 confocal laser scanning microscope (Leica). Twelve-bit images with a 1,024 × 1,024 field size were acquired at 100× oil immersion magnification, and Z-stack images were produced using ImageJ (Fiji).
9
Transcriptional Regulation via ZEB1
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The chicken DF-1 cells and HEK293T cells (obtained from laboratory preservation) were grown in DMEM (GIBCO, Waltham, MA) supplemented with 10% fetal bovine serum (GIBCO, Waltham, MA) in an incubator at 37°C and 5% CO2. According to the instructions, the luciferase reporter vector was transfected into the HEK293T cell line using Lipofectamine 2000 (Thermo Fisher, Waltham, MA). The steps of transfection were as follows: HEK293T cells were grown to a density of 70% in a 24-well plate. Lipofectamine 2000 reagent and pGL3-T, pGL3-C, and pGL3-Basic were diluted in OPTI-MEM(GIBCO, Waltham, MA). Diluted pGL3-T, pGL3-C and pGL3-Basic were added to each tube of diluted Lipofectamine 2000 reagent (1:1) and incubated for 20 min. Finally, the pGL3-T-lipid complex, pGL3-C-lipid complex and pGL3-Basic-lipid complex were added to the cells. After transfection for 48 h, HEK293T cells were collected and treated to measure luciferase activity.
The overexpression vector of ZEB1 pIRES2-EGFP-ZEB1 and the control vector pIRES2-EGFP-basic were transfected into DF-1 cells with Lipofectamine 2000 (Thermo Fisher, Waltham, MA). After transfection for 48 h, DF-1 cells were collected, and RNA was extracted with a cell RNA extraction kit (Tiangen, Beijing, China). The expression levels of ZEB1 and MC1R in pIRES2-EGFP-ZEB1 and pIRES2-EGFP transfected cells were detected by RT‒qPCR.
The overexpression vector of ZEB1 pIRES2-EGFP-ZEB1 and the control vector pIRES2-EGFP-basic were transfected into DF-1 cells with Lipofectamine 2000 (Thermo Fisher, Waltham, MA). After transfection for 48 h, DF-1 cells were collected, and RNA was extracted with a cell RNA extraction kit (Tiangen, Beijing, China). The expression levels of ZEB1 and MC1R in pIRES2-EGFP-ZEB1 and pIRES2-EGFP transfected cells were detected by RT‒qPCR.
10
Modulating hsa_circ_0000729 and miR-1281 in NSCLC
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NCI-H1299 or A549 cells (5×103) were transfected with hsa_circ_0000729 siRNA1 (si-hsa_circ_0000729-1), hsa_circ_0000729 siRNA2 (si-hsa_circ_0000729-2), hsa_circ_0000729 siRNA3 (si-hsa_circ_0000729-3) or negative control (NC; siRNA-ctrl) for 24 h by using Lipofectamine® 2000 (Invitrogen). Hsa_circ_0000729 siRNA1, siRNA2, siRNA3 and siRNA-ctrl were purchased from Genepharma (Shanghai, China). After 24 h of transfection, cells were harvested of use in the subsequent analysis.
For miR-1281 transfection, NSCLC cells were transfected with miR-1281 agomir, miR-1281 antagomir or NC by using Lipofectamine® 2000 (Thermo Fisher Scientific). MiR-1281 agomir, antagomir and NC were obtained from Genepharma. After 24 h of transfection, cells were harvested for use in the subsequent analysis.
For hsa_circ_0000729 overexpression, NCI-H1650 cells were transfected with pcDNA3.1 or pcDNA3.1-hsa_circ_0000729 for 24 h by using Lipofectamine® 2000 (Thermo Fisher Scientific). pcDNA3.1 and pcDNA3.1-hsa_circ_0000729 were obtained from Genepharma.
For miR-1281 transfection, NSCLC cells were transfected with miR-1281 agomir, miR-1281 antagomir or NC by using Lipofectamine® 2000 (Thermo Fisher Scientific). MiR-1281 agomir, antagomir and NC were obtained from Genepharma. After 24 h of transfection, cells were harvested for use in the subsequent analysis.
For hsa_circ_0000729 overexpression, NCI-H1650 cells were transfected with pcDNA3.1 or pcDNA3.1-hsa_circ_0000729 for 24 h by using Lipofectamine® 2000 (Thermo Fisher Scientific). pcDNA3.1 and pcDNA3.1-hsa_circ_0000729 were obtained from Genepharma.
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