Cells were plated into 12-well plates (1 × 105 cells/well for HEK293 cells, 2 × 105 cells/well for SW620 and NCI-H460 cells and 4 × 105 cells/well for Panc10.05 cells) and incubated at 37 °C, 5% CO2 for 24 h before transfection with a 4:1 DNA ratio of pCMV6-Affimer-tGFP and FLAG-ERK plasmids using Lipofectamine 2000 (SW620, HEK293, NCI-H460) or X-tremeGENE 9 (Roche; Panc10.05). After 24 h cells were serum-starved for 1 h, HEK293 cells were then stimulated with 25 ng/ml EGF (Gibco) for 5 min (other cell lines were not stimulated). Cells were washed with ice-cold DPBS then incubated for 10 min on ice with NP-40 lysis buffer. Cleared lysates were incubated overnight at 4 °C with 20 μl anti-FLAG M2 magnetic beads (SigmaAldrich). The beads were washed 3× with TBS before protein elution by incubation at 95 °C for 5 min in SDS sample buffer. Levels of ERK and pERK were then analyzed by immunoblotting with anti-ERK antibody (1:2000, Abcam, ab184699) and phospho-ERK antibody (1:1000, Abcam, ab76299). Densitometry analysis used ImageJ software v.1.52 (NIH, Maryland).
Lipofectamine 2000
Manufactured by Roche
Sourced in Germany, United States, China
Lipofectamine 2000 is a transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, mRNA, or siRNA, into a variety of cell types. It facilitates the uptake and expression of the delivered genetic material within the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Peg it, by System Biosciences (6 mentions)
X tremegene 9, by Roche (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lentivirus qpcr titer kit, by Applied Biological Materials (2 mentions)
Ab76299, by Abcam (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Ab184699, by Abcam (1 mentions)
Cell lysis reagent, by Promega (1 mentions)
Steady glo substrate, by Promega (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Opti mem, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Santa Cruz Biotechnology (1 mentions)
Opti mem reduced serum media, by Thermo Fisher Scientific (1 mentions)
Celastrol, by Merck Group (1 mentions)
Amplitaq dna polymerase, by Roche (1 mentions)
Pc 9 cells, by American Type Culture Collection (1 mentions)
Tri reagent, by Solgent (1 mentions)
Brdu assay kit, by Roche (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
17 protocols using lipofectamine 2000
1
Affimer-ERK Interaction Profiling
2
Transfection and Silencing Assays in Cell Lines
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HEK293T cells were co-transfected with reporter vectors along with other expression vectors or molecular clones using Lipofectamine 2000 (Invitrogen, USA) and harvested 36 h post-transfection for luciferase assay. The cells were lysed in cell lysis reagent (Promega, USA) and luciferase assays were performed using Steady-Glo substrate (Promega, USA) as described earlier (30 (link)). Jurkat cells were transfected using x-treme gene HP DNA transfection reagent (Roche Applied Bioscience, Germany).
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
For silencing studies, cells were first transfected with siRNA using Lipofectamine 2000 as per the manufacturer's instructions, followed by second transfection (reporter plasmid/ expression vectors) or infection as described earlier (52 (link)). Cells were harvested 48 h post-transfection/infection. Knockdown was confirmed by immunoblotting with respective antibodies. Immunoblotting with GAPDH served as the loading control.
3
Stable UCP2 Overexpression and Silencing
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UCP2 overexpression experiments were performed using a pcDNA3.1+ expression vector containing the rat cDNA of UCP2 that we designed previously. The cells were transfected with 5 μg of either the UCP2-bearing vector or control vector with Lipofectamine 2000 (Roche Diagnostic) following the manufacturer's recommendation. Eight hours after transfection, cells were selected using G418 sulphate (600 μg/mL) for 21 days. The cell colonies resistant to G418 were harvested. Stably expressing UCP2-transfected cells were cultured for further studies. UCP2 silencing experiments were carried out with specific small interfering (si) (5′-GUGGUCAAGACGAGAUAUATTUAUAUCUGUCUUGACCACTT-3′) RNA targeting UCP2 mRNA and a nontargeting (NT) siRNA (5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′) purchased from Invitrogen Technologies (Shang Hai, China). Cells were transiently transfected with siRNA according to the manufacturer's protocol (Invitrogen Technologies).
4
Transfection and RNAi Assay Protocol
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Transfection was performed using Lipofectamine 2000 or X-treme gene HP DNA transfer reagent (Roche, Shanghai, China) and opti-MEM reduced serum media (Life Technologies - Invitrogen, Shanghai, China) according to the manufacturers' instruction. For RNAi assay, small interference RNAs (siRNAs), including sifilamin C-1, 2, 3 and a negative control (siNC) (Supplementary Table 1 ), were purchased from Shanghai GenePharma Co., Ltd. For transfection, 120 pmol (GenePharma, Shanghai, China) or 100 pmol (Santa Cruz, Shanghai, China) siRNAs were used to transfect GC cell lines using Lipofectamine 2000 and opti-MEM.
5
Celastrol Inhibits Axl Signaling in PC-9 Cells
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Reagents and antibodies. Celastrol was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). PC-9 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Primers for Axl were synthesized by the domestic company, Bioneer Corp. (Daejeon, Korea). TRI reagent was obtained from Solgent Co., Ltd. (Daejeon, Korea). AmpliTaq DNA polymerase and Lipofectamine 2000 were obtained from Roche Diagnostics Corp. (Indianapolis, IN, USA) and Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA), respectively. For Western blot analysis, specific antibodies against Axl and GAPDH, as well as secondary antibodies were obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA).
6
RASAL1 Effects on HepG2 Proliferation
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To measure the effect of RASAL1 on proliferation activity, 3×103 cells/well HepG2 cells were plated onto 96-well plates. Following overnight culture at 37°C, HepG2 cells were transfected with 100 ng/well pcDNA3.1 or RASAL1 using Lipofectamine® 2000 at 37°C, and after 24 h of incubation at 37°C, cell proliferation was measured with a BrdU assay kit (Roche Applied Science, Penzberg, Germany) in accordance with the manufacturer's protocol. All experiments were repeated three times independently.
7
BMP9 Transfection in MG63 Cells
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To investigate the role of BMP9 in cell function, MG63 cells and homogenized cells from patient tissue were transected either with pcDNA3.1 or pcDNA3.1-BMP9 plasmids (Shanghai Blue Gene Biotech, China) and Lipofectamine 2000 (Roche, Switzerland). The transfection procedures were performed by following the manufacturer instructions. In brief, a given amount (e.g., 2 µg) of pcDNA (pcDNA3.1 or pcDNA3.1-BMP9) was mixed with 9 µL cell culture medium and 5 µL Lipofectamine 2000. The mixture was added into cells cultured in cell culture plates (1 x 10 5 cells per well) followed by culturing at 37°C and 5% CO 2 for 24 or 48 h.
8
CXCR6 Silencing in NSCLC Cells
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For plasmid transfections, NSCLC cells were transfected with vectors of CXCR6-siRNA with the following target sequences: 5ʹ-ACGGATGTGTTCCTGGTGA −3ʹ or negative control RNA according to the manufacturer’s instructions using Lipofectamine 2000 (Roche, USA). Briefly, prior to treatment, Lipofectamine 2000/siRNA complexes were produced in reduced serum medium, OptiMEM (Invitrogen) at the recommended ratio of 20 pmol siRNA per 1ul Lipofectamine 2000. NSCLC cells were then treated with Lipofectamine 2000/siRNA complexes for 6 h before being replaced with RPMI 1640 containing 10% fetal bovine serum. NSCLC cells were also treated with Lipofectamine 2000/NC siRNA complexes as above descried.
9
Efficient Knockdown of AMPK-α1 in Cells
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The AMPK-α1 siRNA(−1) (5′-GCAUAUGCUGC AGGUAGAU-3′), the AMPK-α1 siRNA(−2) (5′-AAGG AAAGTGAAGGTGGGCAA-3′) and the scramble control siRNA were all provided by Dr. Biao Xu's Lab [32 (link)]. Transient transfection was performed by the Lipofectamine 2000 reagent (Roche) according to the manufacturer's instructions. Transfection efficiency was determined by Western blots in resulting cells.
10
Overexpression and Knockdown of IMP3 in Cancer Cells
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IMP3 cDNA (TrueClone Full-Length cDNA clone, Origene) was amplified by PCR, inserted in the XhoI and Sac II sites of pLenti6/V5 plasmid, and transfected to the 293T cells to obtain the virus. Then the Caki-1 cells were transduced with the virus. Two days post-transduction, cells were cultured in selection medium with 5 μg/ml blasticidin until untransducted cells died.
Achn cells were transfected with control shRNA or IMP3 shRNA plasmids separately. The vector structures were described by An et al [16 (link)], the sequence of IMP3 shRNA was listed inS2 Table . For transfection, cells were plated in 6-well plates (1.5 ×105 cells/well) 24 h before and transfected with Lipofectamine 2000 (Roche Applied Science) according to the manufacturer’s instructions. Two days post-transfection, cells were cultured in selective medium with the addition of 800 μg/ml Geneticin (Invitrogen) until untransfected cells died.
Achn cells were transfected with control shRNA or IMP3 shRNA plasmids separately. The vector structures were described by An et al [16 (link)], the sequence of IMP3 shRNA was listed in
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