A LINC00472 transcript (2933 bp, NR_026807.1) was assembled and inserted in a lentiviral vector, pCDH-EF1-MCS-pA-PGK-copGFP-T2A-Puro (pCDH), as previously described (13 (link)). The sequence of the insert has been confirmed by sequencing. MB231 and Hs578T cells were transfected with the LINC00472 plasmid or an empty plasmid (pCDH vector only) using the Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Cells with stable expression of LINC00472 were selected through puromycin screening (Thermo Fisher Scientific). To maintain stably transfected cells, puromycin was added into culture medium, and the puromycin-containing culture medium was replaced every 3 days. A single cell clone was also generated from the stable cell pool through the limiting dilution cloning. Plasmids (pCMV-ESR1) with and without the full-length of human ESR1 (NM_000125, #RC213277) and (pCMV-vector, #PS100001), respectively, were purchased from Origene Technologies, and the plasmids were transfected into the 293T cells and breast cancer cell lines using the Lipofectamine 3000 reagent (Themo Fisher Scientific).
Lipofectamine 3000 reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Canada, Japan, Denmark, Australia, France, Italy, Switzerland
Lipofectamine 3000 reagent is a transfection reagent used to facilitate the delivery of nucleic acids, such as plasmid DNA, into mammalian cells. It is designed to improve transfection efficiency and cell viability.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (418 mentions)
Dual luciferase reporter assay system, by Promega (247 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (174 mentions)
Opti mem, by Thermo Fisher Scientific (167 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (134 mentions)
Dual luciferase reporter assay kit, by Promega (99 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (99 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (88 mentions)
Si nc, by GenePharma (85 mentions)
Opti mem medium, by Thermo Fisher Scientific (76 mentions)
Streptomycin, by Thermo Fisher Scientific (71 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (70 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (68 mentions)
Puromycin, by Merck Group (64 mentions)
Polybrene, by Merck Group (62 mentions)
Penicillin, by Thermo Fisher Scientific (59 mentions)
Sirna, by GenePharma (58 mentions)
Mir nc, by GenePharma (49 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (45 mentions)
Pcdna3, by Thermo Fisher Scientific (45 mentions)
4 180 protocols using lipofectamine 3000 reagent
1
Overexpression of LINC00472 in Breast Cancer
2
Stable Cell Line Generation via Transfection
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350,000 cells were plated in a p6-well dish and transfected with 1 μg DNA using Lipofectamine3000 reagent (Thermo Scientific). Next day, media was replenished and selected using 1 mg/ml G418 antibiotic until no cells were remaining in non-transfected control. Plasmid transfections were performed using Lipofectamine 3000 reagent (Thermo Scientific) according to manufacturer’s protocol. For the experimental analysis, one clone for each variant was used in multiple cell lines. The clones were chosen based on the relative levels of HA-tag and DVL-1 levels.
3
Modulating NLRP3 Expression in Esophageal Cancer
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To knockdown NLRP3, KYSE-70 and TE13 cells were transfected with NLRP3-small interfering RNA (siRNA) and a non-silencing siRNA (Guangzhou RiboBio Co., Ltd.), as negative control. For RNA interference, 50 pmol siRNA was transiently transfected into the cells using 3.75 µl Lipofectamine™ 3000 Reagent (Thermo Fisher Scientific, Inc.) and then cells were incubated for 48 h.
To overexpress NLRP3, KYSE-510 and EC9706 cells were transfected with the plasmid pCDNA3.1 (+)-NLRP3 and pcDNA-3.1(+) vector plasmid as negative control. Cells were then incubated for 18 h, and then transfection was conducted with Lipofectamine™ 3000 Reagent and P3000™ reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.
To overexpress NLRP3, KYSE-510 and EC9706 cells were transfected with the plasmid pCDNA3.1 (+)-NLRP3 and pcDNA-3.1(+) vector plasmid as negative control. Cells were then incubated for 18 h, and then transfection was conducted with Lipofectamine™ 3000 Reagent and P3000™ reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.
4
Immunofluorescence assay in glioma cells
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LN229 and U251 cells were seeded in 8 wells and transfected with plasmids using Lipofectamine™ 3000 reagent (Thermo Fisher Scientific; USA) according to the manufacturer’s protocol, and the control groups were only treated with Lipofectamine™ 3000 reagent (Thermo Fisher Scientific; USA). Cells were fixed with 4% paraformaldehyde for 15 min and incubated with 0.1% Triton X-100 for 10 min. Then, the cells were incubated with 5% BSA for 30 min at room temperature and incubated with primary antibody at 4 °C overnight. After 3 washes with PBS, the cells were incubated with fluorescence secondary antibody (DyLight 488, Thermo Fisher) for 1 h and then stained with 1 U/mL phalloidin (CA1670, Solarbio) for 20 min. A LeicaSP8 confocal microscope (Lecia Microsystems, Wetzlar, Germany) was used to capture images, and ImageJ was used to analyze the data.
5
Luciferase Assay for HDR Evaluation
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Luciferase reporter DNA plasmids for HDR were digested with I-SceI (New England Biolabs, Inc. #R0694) according to I-SceI manufacturer instructions. Digestion was confirmed on a 0.7% agarose gel and then purified using the QIAquick PCR Purification Kit (QIAGEN # 28104). Primary human skin fibroblasts were seeded at 1 × 105 cells per well in a 12-well dish the day before transfection. For experimental conditions, 4 μg of the digested plasmid and 50 ng of a Ranilla plasmid were transfected with Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to manufacturer instructions. For control conditions, 2 μg of a control plasmid and 50 ng of a Renilla plasmid were transfected with Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to manufacturer instructions. Cells were collected and lysed in 1× Passive Lysis Buffer (Promega) 48 hours after transfection. Lysates were run in triplicate in a 96-well plate and analyzed using the Dual-Luciferase® Reporter Assay System (Promega) and a Synergy HT plate reader (BIO-TEK). The percent reactivation was calculated. Experiments were performed at least three times for biological replicates. Each experiment contained technical triplicates per condition. Statistical significance was determined using the unpaired t-test (GraphPad Prism).
6
Transient Transfection in 24-well Plates
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Transient transfections had ∼75 000 cells in 500 μl of complete supplemented DMEM media added to each well of a VisiPlate-24 well black plate (PerkinElmer, catalog number: 1450-605) and were first grown for 24 h. After 24 h, 500 ng of total plasmid DNA was added to Opti-MEM™ media (Thermo Fisher Scientific, catalog number: 31985062), P3000 reagent and Lipofectamine 3000 reagent (Thermo Fisher Scientific, catalog number: L3000001), outlined in the Lipofectamine 3000 reagent protocol. Transfection solutions were incubated at room temperature for 10 min and added to the cells where mixing occurred by gently shaking. Analysis of all experiments was performed 24–72 h after transfections were completed.
7
Calcium Phosphate Transfection of HEK 293Ts and Lipofectamine 3000 Transfection of S2 Cells
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HEK 293Ts were transfected using the following calcium phosphate method: Per 1 mL of media of the cell culture to be transfected, 50 μL of 2x HeBS28 (link),29 buffer, 1 μg of each DNA construct, and H2O up to 94 μL was mixed. 6 μL of 2.5mM CaCl2 was added after mixing of initial components, incubated for 1:45 minutes at room temperature, and added directly to cell culture. S2 cells were transfected with Lipofectamine 3000 reagent (ThermoFisher) following the manufacturer’s protocol. Transfection mixture contained 10ng/μL of DNA, 1.5% Lipofectamine 3000 reagent, and 2% P3000 reagent, and was brought up to volume with Opti-MEM (ThermoFisher). Transfection mix was incubated for 15 min at room temperature and was then added directly to the S2 cells. 100 μL of transfection mix per 1 mL of cell culture media was used. The transfected cells were imaged 72 hr after the transfection (24 hr after promoter induction).
8
Rescue and Production of CA10 Virus
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To rescue the CA10 virus, 293 T cells seeded at density of 5 × 105 cells / well into a 6-well plate were grown in Opti-MEM® I reduced serum medium (Gibco, Waltham, MA, USA) without antibiotics for 24 h. The co-transfection mixture, which contained 1 μg pSVA-CA10 DNA, 1.5 μg pLVX-Puro-T7 RNA polymerase DNA, 7.5 μL P3000™ Reagent, 10 μL Lipofectamine™ 3000 Reagent and 250 μL Opti-MEM medium per well was prepared according to Lipofectamine™ 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) protocol and inoculated into 293 T cells and incubated for 3 days at 37 °C. The rescued CA10 viruses were harvested by freeze-thawing. The supernatant was then inoculated to RD cells in serum-free DMEM medium. At 24 h post-inoculation, the cells displayed almost 80% cytopathic effect (CPE), suggesting that the co-transfection of pSVA-CA10 DNA with pLVX-Puro-T7 RNA polymerase DNA can produce infectious viruses.
9
RNAi Knockdown Using Lipofectamine
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Small interfering RNAs were purchased from Abgent (San Diego, CA, USA). siRNAs were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.
Short hairpin RNAs were purchased from Abgent. shRNAs were transfected using Lipofectamine 3000 reagent and P3000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Transfected cells were selected with puromycin.
Short hairpin RNAs were purchased from Abgent. shRNAs were transfected using Lipofectamine 3000 reagent and P3000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Transfected cells were selected with puromycin.
10
Calcium Phosphate Transfection of HEK 293Ts and Lipofectamine 3000 Transfection of S2 Cells
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