Both ccRCC cells A498 and 786O cells line were transfected with miR-187-3p mimics (Cat. No. miR11272492907-1-5, RiboBio, Guangzhou, China), miR-187-3p inhibitor (Cat. No. miR20000262-1-5, RiboBio), negative control (NC, RiboBio) and LRFN1 overexpression plasmid using Lipofectamine™ 3000 reagent (Cat. No. L3000001, Invitrogen) according to the manufacturer’s instructions. Both A-498 and 786-O cells were transfected with 50 nM miRNAs, 3 µmol/l siRNAs, and 1 µg/µl overexpression plasmid (pcDNA3.1-LRFN1 from Shanghai GeneChem Co., Ltd.) using Lipofectamine™ 3000 reagent (Cat. No. L3000001, Invitrogen) according to the protocols. Cells were harvested for further investigations after transfection for 24 h.
Lipofectamine 3000 reagent
Manufactured by RiboBio
Sourced in China
Lipofectamine 3000 reagent is a cationic lipid-based transfection reagent used for the delivery of DNA, RNA, and other macromolecules into a variety of cell types. It facilitates the uptake of these molecules into the cells by forming complexes that can be readily internalized.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Genechem (1 mentions)
Nc mimics inhibitor, by RiboBio (1 mentions)
Pmirglo, by RiboBio (1 mentions)
Pmirglo, by Promega (1 mentions)
293t cell, by Thermo Fisher Scientific (1 mentions)
Small interfering si rna, by RiboBio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Sirna, by RiboBio (1 mentions)
Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions)
Agomir nc, by RiboBio (1 mentions)
Si nc, by RiboBio (1 mentions)
Site directed mutagenesis kit, by New England Biolabs (1 mentions)
Pmir rb report vector, by RiboBio (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
12 protocols using lipofectamine 3000 reagent
1
Transfection of miR-187-3p and LRFN1 in ccRCC
2
Luciferase Reporter Assay for miRNA Binding
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The sequence of circ-ZNF609 and LRRC1 3’-untranslated region (UTR) containing the predicted miR-432-5p binding site was inserted into a firefly luciferase gene reporter vector pmirGLO (Promega, Madison, USA), respectively. The plasmids were synthesized by Invitrogen. The pmirGLO-circ-ZNF609-WT, pmirGLO-circ-ZNF609-MUT, pmirGLO-LRRC1-WT or pmirGLO-LRRC1-MUT were used with miR-432-5p mimics (inhibitor) or NC mimics (inhibitor) (Ribobio) were co-transfected into tumor cells with Lipofectamine 3000 reagent. After 48 h transfection, according to the manufacturer’s guidelines, luciferase was detected though using the dual luciferase reporter detection system kit (Promega).
3
USP8 Silencing in Endothelial Cells
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The expression plasmid containing NICD sequence (aa 1757-2555) was purchased from Genechem (China) and transfected into Ea.hy 926 cells using Lipofectamine 3000 reagents (Invitrogen, USA). Stable clones were selected using puromycin at a concentration of 2 μg/mL for two weeks. siRNA targeting USP8 and scramble control siRNA were purchased from RiboBio (China) and transfected using Lipofectamine 3000 reagent. The siRNA sequences were: si-USP8 1# (human) (5’-CTGGAACCTTTCGTTATTA-3’), si-USP8 2# (human) (5’- TCATCTCGATGACTTTAAA-3’), and si-USP8 3# (human) (5’- CTACGATGGCAGGTGGAAA-3’). The efficiency of si-USP8 transfection was evaluated using western blot analysis.
4
Macrophage Polarization via Exosomes
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MH-S cells (ZQ0921) and 293T cells (ZQ0033) were obtained from Cell Research (China). MH-S cells were cultured in RPMI-1640 (Gibco, USA) with 10% FBS, 0.05mM β-Mer (Gibco, USA), and 1% PS. 293T cells were cultured in DMEM (high glucose, Gibco, USA) with 10% FBS (Gibco, USA) and 1% PS. Cells were maintained under 37 °C with 5% CO2. For cell transfection, miR-7 mimic (5′-UGUUGUUUUAGUGAUCAGAAGGU-3′; 5′-ACCUUCUGAUCACUAAAACAACA-3′), miR-7 inhibitor (5′-ACCUUCUGAUCACUAAAACAACA-3′), si-PIM1 (5′-GATCATCAAGGGCCAAGTGTT-3′), and the related negative control were transfected using lipofectamine 3000 reagent (Ribobio, China) according to the manufacturer's illustration. Serumal exosomes (10 μg/ml) were incubated with cells for 36 h to examine the role of exosomes in macrophage polarization.
5
SREBF1 and ApoM Silencing in Liver Cells
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Huh-7 cells (BeNa Culture Collection) and Mhcc97h cells (Guangzhou Saiku) were cultured in high-sugar DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Shanghai ExCell Biology, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and were incubated at 37°C in an atmosphere with 5% CO2. As for the reason for choosing Huh7 and Mhcc97h cells, it was observed that SREBF1 was highly expressed in Huh7 and Mhcc97h cells (22 (link)). Small interfering (si)RNA (Guangzhou RiboBio Co., Ltd.) was transfected into cells using Lipofectamine® 3000 reagent (cat. no. L3000150; Thermo Fisher Scientific, Inc.). The sequences were as follows: si-negative control (NC), 5′-GGCTCTAGAAAAGCCTATGC-3′ (this control was non-targeting), si-SREBF1, 5′-CGGAGAAGCTGCCTATCAA-3′ and si-ApoM, 5′-GAGCACAGATCTCAGAACT-3′.
6
Mitochondrial Dynamics Regulation in BMDCs
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BMDCs were incubated in FBS-free medium for 2 hours after 6 days of culture. Then, the cells were transfected with MFN1 siRNA (sense, 5′-GACGACCCGTGCGAAAGA-3′ and antisense, 5′-AGCTTCTCGGTTGCATAGGGGACA-3′) or nonspecific control siRNA (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-AAGCCUAGUUCAAAGAUGGTT-3′; RiboBio, Guangzhou, China) (2 μl, 20 μM) using 4 μl of Lipofectamine 3000 reagent in 1640 medium with 10% FBS. After being transfected for 48 h, the efficiency of MFN1 silencing was determined by western blotting.
7
RCC Cell Line Transfection Protocol
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The human ccRCC cell line, 786-O, was obtained from the National Collection of Authenticated Cell Cultures of China (No. TCHu186). The 786-O cells were cultured in culture medium RPMI-1640 (C11875500BT, GIBCO by Life Technologies, USA), supplemented with 10% fetal bovine serum (Hyclone, Life Sciences, Shanghai, China). Cells were incubated in a humidified atmosphere incubator of 5% CO2 at 37°C. 786-O cells have been transfected with double-stranded siRNA according to the protocol of the manufacturer using plasmid using Lipofectamine 3000 reagent (RiboBio) in a six-well plate, as previously described (17 (link)). The transfection dose for each well was 15 μl of siRNA1 (sequences: GGTGCACCTTCATCAACAA), siRNA2 (sequences: GGATTACCAGCGATTTCTA), or non-targeting siRNA (negative control) at a concentration of 10 nmol/L after incubated using RPMI-1640 for 20 min. 786-O cells were harvested for at least 24 h after transfection for further experimental analysis.
8
Colorectal Cancer Cell Lines: Establishment and Transfection
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Colorectal cancer cell lines including HT29, HCT116, LOVO, RKO, SW480, SW620, and T84 were purchased from Cell Bank of Chinese Scientific Academy in 2017 and 2018 (Shanghai, China), colon mucosal epithelial cell line-NCM460 was purchased from INCELL Company (San Antonio, USA). The above cell lines were cultured in Dulbecco’smodified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Gibco, LifeTechnologies, CA) and 100 μg/ml penicillin–streptomycin. Cells were cultured at 37 °C with5% CO2 in a humidified incubator. siRNAs were purchased from RIBOBIO (Gunagzhou, China), then transfected with Lipofectamine3000reagent. The target sequence of PDCD2L siRNA were as followed: negative control:siN0000001-1–5, si-PDCD2L-1:GCTCAAGAGTGCTAATTTA, si-PDCD2L-2:GGACTATCAGCAGAGAGAA, and si-PDCD2L-3:GGAACAATTCTAGTTTACA.
9
Silencing FSTL1 and Activating miR-124-3p
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Small interfering RNA targeting FSTL1 (siFSTL1) was synthesized by Guangzhou RiboBio Co., Ltd., and a scrambled siRNA (siNC; Guangzhou RiboBio Co., Ltd.) was used as the negative control. One day before transfection, cells were digested with 0.25% trypsin (cat. no. C0202; Beyotime Institute of Biotechnology). Then, 50 µl of Opti-MEM® medium (cat. no. 31985062; Invitrogen; Thermo Fisher Scientific, Inc.) was mixed with siFSTL1(cat. no. siB14715103107-1-5; Guangzhou RiboBio Co., Ltd.) or siNC. And 3 µl Lipofectamine® 3000 reagent (cat. no. L3000015; Thermo Fisher Scientific, Inc.) was diluted with 50 µl OptiMEM. Then, the above two mixtures were mixed. Then cells were transfected with the mixture according to the Lipofectamine® 3000 kit (Thermo Fisher Scientific, Inc.).
miR-124-3p agomir (miR40000422-4-5 Guangzhou RiboBio Co., Ltd.) and negative control (agomir NC) were synthesized by Guangzhou RiboBio Co., Ltd. Cells were seeded in six-well plates (1x105 cells/ml) and transfected with 50 nM miR-124-3p agomir or 50 nM agomir NC using Lipofectamine® 3000 kit (Thermo Fisher Scientific, Inc.). The transfection efficiency was observed by a fluorescence microscope after 24 h.
miR-124-3p agomir (miR40000422-4-5 Guangzhou RiboBio Co., Ltd.) and negative control (agomir NC) were synthesized by Guangzhou RiboBio Co., Ltd. Cells were seeded in six-well plates (1x105 cells/ml) and transfected with 50 nM miR-124-3p agomir or 50 nM agomir NC using Lipofectamine® 3000 kit (Thermo Fisher Scientific, Inc.). The transfection efficiency was observed by a fluorescence microscope after 24 h.
10
Deciphering ACSL4 and lncAABR07025387.1 Interactions
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ACSL4 and lncAABR07025387.1 3′-UTRs were amplified in pmiR-RB-REPORT vectors (Ribobio). The binding sites were mutated using a site-directed mutagenesis kit (NEB E0554, United States). HEK293T cells were plated into six-well plates at a density of 106 cells per well and co-transfected with a reporter containing the wild-type or mutant plasmid and miR-205 mimic, inhibitor, or related control using Lipofectamine 3000 reagent (Ribobio). After 48 h, luciferase activity was detected using a Dual-Luciferase Assay System (Promega Corp., Madison, WI, United States) according to the manufacturer’s guidelines.
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