Drosophila S2 cells (Invitrogen) were cultured at 25° C in Schneider’s Drosophila medium (GIBCO) supplemented with 5% fetal calf serum, 100 U/ml penicillin and 100 ug/ml streptomycin. S2 cells were seeded at 2.5 x104 cells per well of 24-well plates and grown for 24 hours before transfection. Transient transfections were performed using Effectene reagent (QIAGEN) according to manufacturer’s instructions. Ago-1, Ago-2 and luciferase double-stranded RNAs were generated as described in Carré et al., 2013. For RNAi knockdown experiments, 3 μg of dsRNA were co-transfected with the appropriate DNA plasmids using Effectene reagent (QIAGEN) and expression of the reporter plasmids was triggered 24 hours later by addition of 500 μM CuSO4. After 48 additional hours, cells were harvested for western blot analyses.
Effectene reagent
Manufactured by Qiagen
Sourced in United States, Germany, China
Effectene reagent is a transfection reagent designed for efficient delivery of nucleic acids into eukaryotic cells. It facilitates the uptake of DNA, RNA, or other macromolecules into cells, enabling various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (9 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Polybrene, by Merck Group (6 mentions)
Hek293t, by American Type Culture Collection (6 mentions)
Mzip000 pa 1, by System Biosciences (6 mentions)
Pmirh146apa 2, by System Biosciences (6 mentions)
Anti flag, by Merck Group (5 mentions)
Nocodazole, by Merck Group (5 mentions)
Mg132, by Merck Group (5 mentions)
Glutamine, by Thermo Fisher Scientific (5 mentions)
Schneider s medium, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
205 protocols using effectene reagent
1
Drosophila S2 Cell RNAi and Transfection
2
Silencing Gαs and PKA Cα via siRNA Transfection
3
Akt/S473A Protein Expression and Purification
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Akt/S473A protein was expressed and purified from baculovirus-infected HepG2 cells. Briefly, HepG2 cells were maintained in Trypsin-EDTA media (Invitrogen) and infected with baculovirus encoding pEX-3-Akt for 24 h. The infected cells were treated with 10% FBS for 15 min prior to lysis. The infected cells were lysed in PBS containing 1% Triton X-100 and 10 mM imidazole. Transient transfection of HepG2 cell was carried out by using Effectene reagents (Qiagen). Lipofectamine 2000 (Invitrogen) was used to transfect Akt mut into HepG2 cells.
4
Silencing Gαs and PKA Cα via siRNA Transfection
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For RNA interference experiments, control (Dharmacon siGLO RISC-free control siRNA), Gαs, or PKA Cα siRNAs (Dharmacon) were transfected into cells using a calcium phosphate method. Briefly, 2.5M CaCl2 was added to siRNA (5μM) diluted in 2X HBS. The transfection mixture was mixed vigorously, incubated for 20 min at room temperature and added dropwise to plate containing 50% confluent cells. Cells were plated on CL 96 hrs post-transfection and experiments were performed. The following siRNA sequences were used in this study: Gαs: 5′-CCACCAAAGUGCAGGACAUUU-3′; PKA Cα: 5′-GAGUAAAGGCUACAACAAAUU -3′. For GFP-PH experiments, cells were seeded at 50% confluence and transfected with 2.5 μg of the GFP-PH construct using Effectene reagents (QIAGEN) according to the manufacturer’s protocol. Experiments were performed 48 hr after transfection.
5
Immunoprecipitation of Myc- or Flag-tagged Proteins from Drosophila S2 Cells
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Drosophila S2 cells were cultured in M3 media (Sigma) with 10% Insect medium supplement (Sigma). Transfection was carried out with Effectene reagent (Qiagen) according to manufacturer’s instructions. Total of 1–2 μg DNA was used for each transfection. For immunoprecipitation, cells were lysed in 0.1% CHAPS buffer, and the lysates were precleared by incubating with protein G-sepharose beads (Roche) for 1 h at 4 °C. The G-sepharose beads were immunoprecipitated with anti-Myc (Abcam) or anti-Flag (Sigma) at 4 °C for 1 h. The immunoprecipitates captured by protein G-sepharose were incubated with the clear lysates overnight at 4 °C. Immunoprecipitates were washed three times in cold IP buffer. The samples were boiled in protein loading buffer at 94 °C for 5 min and then subjected to SDS-PAGE. Western blot was performed with mouse Flag (Sigma) or mouse V5 at 1:5000 (Invitrogen).
6
Overexpression of TRIM37 Protein
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A TRIM37 cDNA clone (Origene Technologies) was subcloned into the vector p3X-FLAG-Myc-CMV-26 (Sigma) using Not1 and Xba1 sites, and verified by full-length sequencing. The TRIM37 expression vector was transfected into cells using Effectene reagent (Qiagen) and stable clones were selected. Over-expression of TRIM37 was confirmed by immunoblotting using TRIM37 (21st Century) and anti-FLAG (Sigma M2) antibodies.
7
Overexpression of TRIM37 Protein
8
Cell Culture and Transfection Protocol
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HeLa CCL-2, HEK293, NIH3T3 and COS-1 cells were obtained from ATCC and grown in high-glucose DMEM medium (HyClone) containing 10% (v/v) FBS (Gemini Bio-Products), 0.5% (v/v) penicillin–streptomycin (Mediatech). All cell lines were grown at 37 °C in a humidified 5% CO2 atmosphere. Transfection of plasmids was performed with an Effectene reagent (Qiagen) according to the manufacturer’s protocol. Cells were not tested for mycoplasma contamination or cross-contamination.
9
Plasmid Expression of Near-infrared Fluorescent Proteins
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The plasmids used for expression of iRFP670 and iRFP720 in mammalian cells were based on pEGFP-N1 backbone (Clontech). The iRFP670 and iRFP720 genes were PCR-amplified as AgeI-NotI fragments and swapped with the EGFP gene in pEGFP-N1 vector, thus generating the piRFP670 and piRFP720 plasmids.
A rat adenocarcinoma MTLn3 cell line was grown in αMEM medium (Life Technologies) containing 5% fetal bovine serum, 0.5% penicillin-streptomycin, and 2 mM glutamine. Plasmid transfections were performed using an Effectene reagent (Qiagen) according to the manufacturer's protocol. Stably expressing preclonal cell mixtures were selected with 700 μg/ml G418 antibiotic. Sorting of fluorescent cells was performed using a MoFlo XDP sorter (Beckman Coulter) equipped with a 676 nm Kr laser and a 700 nm long-pass emission filter.
A rat adenocarcinoma MTLn3 cell line was grown in αMEM medium (Life Technologies) containing 5% fetal bovine serum, 0.5% penicillin-streptomycin, and 2 mM glutamine. Plasmid transfections were performed using an Effectene reagent (Qiagen) according to the manufacturer's protocol. Stably expressing preclonal cell mixtures were selected with 700 μg/ml G418 antibiotic. Sorting of fluorescent cells was performed using a MoFlo XDP sorter (Beckman Coulter) equipped with a 676 nm Kr laser and a 700 nm long-pass emission filter.
10
Cell Culture and Transfection Protocols
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Cell culture. HDF cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and supplemented with 10% fetal bovine serum, 5% minimum essential medium nonessential amino acids (100×, Gibco), 5% penicillin and streptomycin (Gibco), and l -glutamine (Gibco). T-Rex-CHO cells were grown in Ham’s F12K medium (American Type Culture Collection) with the same supplements. Drosophila S2 cells were cultured in Express Five SFM Medium (Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) (Gibco), and 45 ml of 200 mM l -glutamine (Gibco) per 500 ml of medium.
Plasmids and mRNA were introduced to the cells by the Neon Transfection System (Invitrogen) with 100-μl tips according to cell-specific protocols (www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html ). Cells electroporated with DNA plasmids were harvested after 48 hours if not indicated differently. Cells electroporated with mRNA were harvested after 4 hours, if not indicated differently. All transfections in S2 cells were performed using Effectene reagent (Qiagen).
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Plasmids and mRNA were introduced to the cells by the Neon Transfection System (Invitrogen) with 100-μl tips according to cell-specific protocols (
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