For each well of the 6-well plates, 3×105 cells were plated in fresh media without antibiotics for 24 h prior to transfection. Cells were transfected with either 2 mg pcDNA3.1 vector containing GLI1 cDNA or pcDNA3.1 vector control (Thermo Fisher Scientific, Inc.) using Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For RNA interference, cells in 6-well plates were transfected with 100 pmole of synthesized GLI1 small interfering (si)RNA (siRNA-1, Assay ID 107670; siRNA-2, Assay ID 115641, Thermo Fisher Scientific, Inc.) or scrambled siRNA using Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After the transfection, the cells were cultured for 48 h before further analysis with drug treatment.
Lipofectamine 2000 transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Canada, Austria, Japan, Netherlands, Sweden, Spain, France, Switzerland, Italy, Lithuania
Lipofectamine 2000 is a cationic lipid-based transfection reagent used for the introduction of nucleic acids, such as DNA and RNA, into a variety of eukaryotic cell types. It facilitates the uptake and delivery of these molecules into the cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (339 mentions)
Opti mem, by Thermo Fisher Scientific (250 mentions)
Dual luciferase reporter assay system, by Promega (205 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (153 mentions)
Streptomycin, by Thermo Fisher Scientific (91 mentions)
Penicillin, by Thermo Fisher Scientific (88 mentions)
Opti mem medium, by Thermo Fisher Scientific (84 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (82 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (79 mentions)
Polybrene, by Merck Group (64 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (57 mentions)
Puromycin, by Merck Group (57 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (52 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (51 mentions)
Dual luciferase reporter assay kit, by Promega (44 mentions)
Trizol reagent, by Thermo Fisher Scientific (43 mentions)
Glutamax, by Thermo Fisher Scientific (42 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (41 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (38 mentions)
Pspax2, by Addgene (38 mentions)
3 831 protocols using lipofectamine 2000 transfection reagent
1
GLI1 Overexpression and Knockdown
2
Validation of miR-107 Target Genes
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To generate pmiR-RB-Report™-hDICER1 and pmiR-RB-Report™-hPTEN dual luciferase reporter plasmids, 3ʹUTRs of DICER1 and PTEN were PCR-amplified using HEK293T genomic DNA as a template and cloned into the pmiR-RB-REPORT™ dual luciferase reporter vector (RiuboBio Co. Ltd., Guangzhou, China.). To generate reporter plasmids with mutated miR-107 binding sequences, site-directed mutagenesis was performed to mutate “ATGCTGC” to “TACGACG” on hDICER1 3ʹUTRs, and “ATGCTGC” to “TACGACG” on PTEN 3ʹUTR. To validate the target genes of hsa-miR-107, HEK293T cells were co-transfected with hsa-miR-107 mimic (50 nM), together with pmiR-RB-Report™-hDICER1 or pmiR-RB-Report™-hPTEN plasmids (1 μg) using Lipofectamine 2000® Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were then cultured for 24 hrs before being analyzed for reporter expression using the Dual-Luciferase Reporter Assay System (Promage corporation, Wisconsin, USA ).
To knock down DICER1 or PTEN in MDSCs, hDICER1 siRNA (50 nM) or hPTEN siRNA (50 nM) was transfected into HLA-DR−CD33+MDSCs using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific).
To knock down DICER1 or PTEN in MDSCs, hDICER1 siRNA (50 nM) or hPTEN siRNA (50 nM) was transfected into HLA-DR−CD33+MDSCs using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific).
3
Knockdown of Metabolic Enzymes in Cell Lines
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Knockdown of pyruvate carboxylase (PC) (EC number: 6.4.1.1), pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1) (EC number: 1.2.4.1), citrate synthase (CS) (EC number: 2.3.3.16), and lactate dehydrogenase A (LDHA) (EC number: 1.1.1.27) were performed using the specific Invitrogen Silencer® Select siRNAs (PC siRNA ID: s10089, PDHA1 siRNA ID: s10245, CS siRNA ID: s3583, LDHA siRNA ID: s351). Silencer® Select Negative Control siRNA served as negative control. LipofectamineTM 2000 Transfection Reagent (Invitrogen) was used for the transfection of siRNAs. Briefly, cells were seeded into a 60-mm culture dish and were allowed to achieve 95% confluence. 200 pmol siRNA and 6 μL Lipofectamine 2000 Transfection Reagent were incubated in 0.5 mL Gibco Opti-MEM® I Reduced-Serum Medium (Thermo Fisher Scientific) for 5 min, respectively, and then mixed and incubated for 20 min. Cells were transfected with the mixed medium. 6 h after transfection, cells were seeded in 96-well microplates for cell proliferation assay, and in 60 mm culture dishes for RNA and protein extraction.
4
Plasmid Transfection of 293T Cells
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The plasmid sh-DJ1 was a kind gift from Hans-Guido Wendel’s lab at MSKCC, which was purchased from Sigma and constructed by the previously reported methods59 (link). The shRNA plasmids were transfected with Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, cultured 293T cells were washed with Opti-MEM medium (Invitrogen) and then transfected with the shRNA plasmid using Lipofectamine 2000 Transfection Reagent in Opti-MEM medium without serum. 6 h after transfection, the culture medium was replaced with fresh complete DMEM medium. The cells were subjected to further treatment and/or analysis 24 h after transfection.
5
Transient Transfection of miR-200c in Cells
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Transient transfection was performed using Lipofectamine 2000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocols. Briefly, ~1×104 cells were first seeded in cell culture plates. At 50% confluence, cells were transfected with miR-200c mimic or miR-200c inhibitor (cat. nos. 4464066 and 4464084, respectively; Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 Transfection Reagent in RPMI1640/DMEM without serum. Control reactions were simultaneously performed using the miR-200c mimic negative control (NC) or miR-200c inhibitor NC (cat. nos. 4464058 and 4464076, respectively; Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h following transfection, cells were collected for subsequent experiments.
6
Overexpression and Knockdown of Src in Keratinocytes
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Full-length human Src cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from normal skin RNA. The PCR products were digested with NheI and KpnI, and subcloned into the eukaryotic expression vector, pcDNA3.1(+) (GeneCopoeia, Rockville, MD, USA). The recombinant pcDNA3.1(+)-Src plasmid (GeneCopoeia) was confirmed by sequencing. The keratinocytes were plated in 60 cm2 wells for 24 h prior to transfection. Thereafter, the cells were transfected with either 5 µg pcDNA3.1(+)-Src plasmid or the empty vector (GeneCopoeia) using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The siRNA (80 nM; Thermo Fisher Scientific, Waltham, MA, USA) duplex sequence was as follows: sense, 5′CGAGUGCCUUAUCCAAGAATT-3′. The cells transfected with scrambled siRNA (Thermo Fisher Scientific) (sense, 5′UUCUUGGAUAAGGCACUCGTT-3′), which were the mock group, were also transfected using Lipofectamine 2000 transfection reagent. Western blot analysis was performed after an additional 48 h.
7
Overexpression and Knockdown of Tbc1d24, Tldc1, and Tldc2
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For Tbc1d24, Tldc1 and Tldc2 overexpression experiments, human embryonic kidney HEK-293T cells (ATCC® CRL-3216™, American Type Culture Collection (ATCC), Manassas, VA) were transiently transfected with pCDNA3-based plasmids, expressing HA-tagged full-length mouse Tbc1d24, Tldc1 and Tldc2, using Lipofectamine 2000 transfection reagent (ThermoFisher Scientific) according to the manufacturer’s instructions. For Tbc1d24 knockdown experiments, Stealth pre-designed siRNAs (set of three) MSS277591, MSS277592, MSS277593, each targeting three different regions of mouse Tbc1d24 mRNAs and one nontargeting Stealth siRNA (negative control) were purchased from ThermoFisher Scientific. Mouse kidney cortical collecting duct M-1 cells (ATCC® CRL-2038™, ATCC) were transiently co-transfected with a pCDNA3-based plasmid, expressing HA-tagged full-length mouse Tbc1d24, with or without a mixture of all three Tbc1d24 siRNAs (40 nM each) or 120 nM of negative control siRNA using Lipofectamine 2000 transfection reagent (Invitrogen). Seventy-two hours after transfection, cells were lysed and analyzed by western blotting.
8
TTI1 Silencing in CRC Cell Lines
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Human CRC cancer cell lines (CaCO2, HT29) were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). CaCO2 cells were maintained in complete MEM medium with 10% bovine serum albumin, 1% penicillin-streptomycin solution and 1% nonessential amino acid solution. HT29 cells were maintained in McCoy’s 5a complete medium with 10% bovine serum albumin and 1% penicillin-streptomycin solution. Cells were incubated at 37 °C with 5% CO2 and 20% O2. To reduce TTI1 expression, small interfering RNA (siRNA) was used coupled with LipofectamineTM 2000 Transfection Reagent (Invitrogen, USA). siRNA duplexes targeting the TTI1 mRNA were synthesized by Ribobio (Guangzhou, China) as following: GGAAGCAAGTGTGGTGACT. The density of cells reached 30–50% and then cells were transfected with the siRNA targeting TTI1 and negative control siRNA (siCon) through the use of LipofectamineTM 2000 Transfection Reagent (Invitrogen, USA). After a 6-hour transfection, cells were purified for proliferation assay.
9
Transfection of Mouse Testis Cell Lines
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Mouse testis cell lines, GC-1 spermatogonia (from Bioresource Collection and Research Center; BCRC Number: 60312) were grown in Dulbecco's Modified Eagle Medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 1.0 mM sodium pyruvate, and 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. TM3 Leydig cells (from Bioresource Collection and Research Center; BCRC Number: 60475) and TM4 Sertoli cells (from Bioresource Collection and Research Center; BCRC Number: 60254) were grown in Dulbecco's Modified Eagle Medium/F12 with 4.5 g/L glucose, 2.5 mM L-glutamine, 0.5 mM sodium pyruvate, 1.2 g/L sodium bicarbonate, 15 mM HEPES supplemented with 5% horse serum and 2.5% fetal bovine serum at 37°C containing 5% CO2. The mouse testis cell lines, primary testis cells and primary neuron cells were transfected with pIRES-SMN2 minigene-luc plasmid (provided by Dr. Minlei Zhang [33 (link)]) by Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer’s instructions. The primary testis cells were transfected with Tra2-β1 siRNA (Ambion, siRNA ID: s73772) by Lipofectamine 2000 Transfection Reagent. After 48 hours of transfection, total RNA was extracted from transfected cells.
10
Pvt1 Knockdown in C2C12 Myoblasts
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In vitro experiments to down-express Pvt1 were performed by transfecting proliferating C2C12 myoblasts with Lipofectamine 2000 Transfection Reagent (Termo Fisher Scientific) and antisense LNA GapmeRs (Exiqon) (Pvt1_1 ACCGTAGTAGAGTTAA; Pvt1_3 AGTCAACGCTTCACAT). Cells transfected with Lipofectamine 2000 and Antisense LNA GapmeR Negative Controls (Exiqon) were used as negative controls. Silencing efficiency was assessed by qPCR analysis (for extended protocol seeSupplementary Methods ).
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In vitro experiments to down-express Pvt1 were performed by transfecting proliferating C2C12 myoblasts with Lipofectamine 2000 Transfection Reagent (Termo Fisher Scientific) and antisense LNA GapmeRs (Exiqon) (Pvt1_1 ACCGTAGTAGAGTTAA; Pvt1_3 AGTCAACGCTTCACAT). Cells transfected with Lipofectamine 2000 and Antisense LNA GapmeR Negative Controls (Exiqon) were used as negative controls. Silencing efficiency was assessed by qPCR analysis (for extended protocol see
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