Two siRNAs targeting the human Prdx2 gene were designed: siPrdx2-1 (5′-GGAAGTACGTGGTCCTCTT-3′) and siPrdx2-2 (5′-GCCAGATCACTGTTAATGA-3′). The two siRNAs and a control siRNA were synthesized by Sangon Inc. (Shanghai, China). The siRNAs were transfected into HTR8 cells at a final concentration of 100 nmol/l using the Oligofectamine reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Knockdown of c-Myc was performed using specific oligonucleotides purchased from Santa Cruz Biotechnology Inc. (TX, USA), which were transfected into cells using the Oligofectamine reagent (Invitrogen). All cells were cultured for 48 h after transfection before they were harvested for quantitative RT-PCR, western blot and other analyses. To create the c-Myc overexpression construct, the coding region sequence of human c-Myc was cloned into the pEX-2 vector (GenePharma, Shanghai, China). The c-Myc-pEX-2 vector and control vector were purified using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA), and transfected into the cells using Lipofactamine 3000 (Invitrogen) according to the manufacturer’s protocol. All cells were cultured for 48 h after transfection before other assays were performed.
Oligofectamine reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Canada, United Kingdom, Germany, France
Oligofectamine is a transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into eukaryotic cells. It facilitates the uptake of these molecules by the cells, enabling the study of gene expression, gene silencing, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
On targetplus smartpool sirna, by Horizon Discovery (4 mentions)
Opti mem medium, by Thermo Fisher Scientific (4 mentions)
Opti mem 1 reduced serum media, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (3 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (3 mentions)
Sirna, by Qiagen (3 mentions)
On targetplus non targeting pool, by Horizon Discovery (2 mentions)
Huvecs, by Lonza (2 mentions)
X tremegene 9 dna transfection reagent, by Roche (2 mentions)
On targetplus smartpool, by Horizon Discovery (2 mentions)
Sirna pool, by Horizon Discovery (2 mentions)
Sc 37455, by Santa Cruz Biotechnology (2 mentions)
Sc 29827, by Santa Cruz Biotechnology (2 mentions)
L 003506 00, by Horizon Discovery (2 mentions)
Oligonucleotide mix, by Horizon Discovery (2 mentions)
192 protocols using oligofectamine reagent
1
Prdx2 Gene Silencing and c-Myc Modulation
2
Mcl-1 siRNA Transfection Optimization
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Exponentially growing cells were seeded in 6‐well plates (4 × 104/well) and cultured overnight in a 5% CO2 atmosphere at 37°C. The medium was then replaced with Opti‐MEM I Reduced Serum Media (Gibco) containing 20.0 nM Mcl‐1 small interfering RNA (siRNA; GenePharma, Shanghai, China) and oligofectamine reagent (Invitrogen™; Thermofisher Scientific, Waltham, MA, USA) according to manufacturer's recommendations. Forty‐eight hours after transfection, cells were harvested or treated with DMSO vehicle, serial concentrations of ABT‐737, DMC or the combination.
3
Investigating miR-34a's Transcriptional and Apoptotic Effects
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Oligofectamine reagent (Thermo Fisher Scientific, Inc.), miR-34a (cat. no. C-300551-07; GE Healthcare Dharmacon, Inc., Lafayette, CO, USA), b (Assay ID, PM10743; cat. no. AM17100) and c (Assay ID, AM12342; cat. no. AM17000), scrambled mimics (cat. no. 4464058), and antago-scrambled (cat. no. 4464076) and antago-miR-34a miRNAs (Assay ID, MH11030; cat. no. 4464084) (all Thermo Fisher Scientific, Inc.) were employed following the manufacturer's protocol. To detect the effect of miR-34a, b or c on the transcriptional activity of p53, miR-34a, b or c, or negative control scrambled mimics were transfected into HCT116 cells. To detect the effects of miR-34a on apoptosis and cell cycle distribution, antago-miR-34a or antago-scrambled miRNAs were transfected into HCT116 cells. Briefly, 5×105 cells/well were plated in a 6-well plate (Corning Incorporate, Corning, NY, USA) for 16 h. A total of 200 nM microRNA mimics was transfected transiently into cells. Flow cytometry and luciferase reporter assay experiments were performed 24 h after transfection. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were performed 48 h after transfection.
4
Transwell Barrier Integrity Assay
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Transwells™ were obtained from Corning (Schiphol, The Netherlands). Lucifer yellow was purchased from Thermo Fisher. Protease inhibitor cocktail Set III from Calbiochem (Merck, Darmstadt, Germany). Oligofectamine™ Reagent and Pierce BCA Protein Assay were from Thermo Fisher Scientific Inc. (Rockford, IL). For Western blotting and immunofluorescence, antibodies directed against occludin, claudin-5, and ZO-1 were purchased from Invitrogen (Basel, Switzerland), β-actin antibody from Sigma–Aldrich (Buchs, Switzerland), and β-catenin antibody from Chemicon (Millipore, Billerica, MA). Anti-phosphotyrosine antibody agarose conjugate, clone 4G10®, was purchased from Milipore. Secondary antibodies for Western blotting and immunofluorescence were obtained from Jackson ImmunoResearch (Suffolk, UK) or Invitrogen. For the glutathione (GSH) and ROS assay, 5,5-Dithio-Bis-(2-Nitrobenzoic acid) (DTNB) and 2′,7′-Dichlorofluorescin diacetate were obtained from Sigma–Aldrich (Buchs, Switzerland). Isotopic labeled 34S15N-cysteine was designed and synthesized by Dr. T. Sawa (Graduate School of Medical Sciences, Kumamoto University) [54 (link)]. γ-glutamyl cysteine ligase catalytic subunit (GCLc) siRNA (ON-TARGET plus SMART pool) and negative control siRNA (ON-TARGET plus non-targeting pool) were purchased from Dharmacon.
5
CRISPR/Cas9 Knockout and WT HeLa Cell Lines
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Stable CRISPR/Cas9 μ1A KO and WT HeLa cells were a gift from Margaret S. Robinson (University of Cambridge) and have been previously described (20 (link)). H4 (human neuroglioma) cells were obtained from the American Type Culture Collection. PEAK cells are HEK-293T cells transfected with the large T antigen of SV-40 (59 ) and were used for co-immunoprecipitation experiments. To generate HeLa cell lines expressing ER-targeted streptavidin fused to KDEL (ER hook), retrovirus coding streptavidin-KDEL was produced in HEK-293T cells and used to transduce HeLa cells. Transduced cells were selected by incubation in complete media in the presence of 1 μg/ml puromycin. Cells were maintained as previously described (60 ). DNA transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific). The siRNAs were purchased from Dharmacon as nucleotide duplexes with 3′dTdT overhangs, designed to target human γ1 (5′-GGAAGAGCCUAUUCAGGUA-3′) (51 (link)). Transfections of siRNA were performed in two rounds with an interval of 48 h between treatments using Oligofectamine reagent (Thermo Fisher Scientific).
6
Silencing Endothelial Cell Signaling
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Human dermal microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs) were from Lonza (Walkersville, MD). Cells were cultured in endothelial growth medium supplemented according to the manufacturer’s instructions. MyEnd cells (15 (link)) were cultured, as previously described (6 (link)). HMVECs were depleted of Epac1 or Mena/VASP with SMARTpool siRNA oligonucleotides from Dharmacon (Lafayette, CO). Briefly, 100 nmol/L siRNA mix were complexed with OligofectAMINE reagent (Thermo Fisher Scientific, Parsippany, NJ) and cells were exposed to the lipid-oligonucleotide mixture for 4–6 h. Transfection efficiency was evaluated by Western blotting.
7
Transfection and siRNA Knockdown in HCT116 Cells
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p3 × Flag-CMV-importin α1 and p3 × Flag-CMV empty vectors were transfected into HCT116 cells with Lipofectamine 3000 (Thermo Fisher Scientific, Rockford, IL, USA). The sequences of the siRNA (chimeric RNA-DNA) duplexes have previously been described43 (link). Cells were transfected with each siRNA for 48 h using Oligofectamine reagent (Thermo Fisher Scientific).
8
Double-Transfection SIRT1 Knockdown Protocol
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A double-siRNA-transfection approach was used to achieve knockdown. Cells were plated in a 6-well dish the morning of day 1 and allowed to settle on the plates for 3 to 4 h. Cells were then transfected with one of two SIRT1-targeting siRNAs (the sequences are described above in the “Plasmids and compounds” section and were the same as those reported in reference 42 (link)) or with scrambled siRNA (Mission siRNA universal negative-control no. 1 [Sigma]) using Oligofectamine reagent (Thermo) and Opti-MEM (Gibco). For a 6-well dish, 10 μl Oligofectamine and 20 μl Opti-MEM were mixed and the mixture was incubated for 5 min at RT and mixed with 160 μl Opti-MEM and 4 μl 50 μM siRNA. This transfection mixture was incubated for 20 min at RT. Media were removed from cells and replaced with 800 μl Opti-MEM, the transfection mixture was added, and the resulting mixture was incubated at 37°C/5% CO2 for 4 h. After this incubation period, a 1:1 volume of 20% FBS–DMEM was added to the cells. The following day, media were changed for 10% FBS–DMEM. Cells were collected on day 3 and replated for a second round of transfection using the same approach. Cells were then collected on day 5 and prepared for experimental use.
9
Silencing AP-4 and Hook Proteins
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ON-TARGETplus SMART pool siRNAs aimed at the following human targets were purchased from Dharmacon (catalogue number followed by target): L-021474-00-0005, AP-4 ε; L-011918-01-0005, AP-4 μ4; L-016845-01-0005, Hook1; L-020408-02-0005, Hook2; L-013558-01-0005, Hook3; L-014783-02-0005, FHIP (FAM160A2 gene product); L-184739-00-0005, FHIP-L (FAM160A1 gene product). A nontargeting siRNA (cat. D-001810-01-05) was used as control. Cells were seeded on 6-well plates and subjected to two rounds of transfection at times 0 and 48 h with 200 nM siRNAs using the Oligofectamine reagent (ThermoFisher) at a 4 μl/ml concentration. Triple silencing of Hook proteins was carried out at a total siRNA concentration of 480 nM and 7–8 μl/ml Oligofectamine. Cells were split and replated 48–72 h after the second round of treatment and analyzed by IB and IF on the next day.
10
Cell culture and transfection protocol
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HeLa and HEK 293T cells were grown in DMEM supplemented with 10% FCS. PLAT-A packaging cells were grown in DMEM supplemented with 10% FCS, 10 µg/ml blasticidin S, and 1 µg/ml puromycin. Plasmid and siRNA transfection into HeLa cells was performed using X-tremeGENE 9 DNA transfection reagent (Roche) and Oligofectamine reagent (Thermo Fisher Scientific), respectively, according to the manufacturers’ protocols. Plasmid transfection into HEK 293T and PLAT-A packaging cells was performed using polyethylenimine (Polysciences) or Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific).
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