Undifferentiated ST14A cells, a rat striatal progenitor cell line with high transfectability and many neuronal characteristics (gift from Dr. Elena Cattaneo, Milan IT), were cultured according to standard protocols [22 (link)]. 2 μg of total DNA was transfected per well in 6 well plate. JetPEI DNA transfection reagent (Polyplus Transfection Inc.) was used to transiently transfected cells as previously described [23 (link)]. For all transfections unless otherwise noted, cells were imaged 48 hours’ post-transfection, and then harvested for downstream applications. For endogenous α-Syn assay, normal SH-SY5Y cells (ATCC) were transiently transfected using Jet PRIME DNA transfection reagents (Polyplus Transfection Inc.), with control (CON; empty vector) or VH14, VH14PEST, or VH14SCRPEST (scrambled inactive PEST) constructs. They were then differentiated into the neuronal pathways 48 hours post- transfection for 3 days with 10 μM retinoic acid (RA) prior to harvesting [24 (link)]. To verify that targeted degradation is occurring through the proteasome, specific proteasome inhibitor epoxomicin (10 μM per well) or the vehicle DMSO was applied to the cells 12 h prior to the harvest in a subset of experiments.
Jetpei dna transfection reagent
Manufactured by Polyplus Transfection
Sourced in France, United States, United Kingdom
JetPEI is a DNA transfection reagent. It is designed to facilitate the delivery of DNA into cells in culture.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Arnt targeted and non silencing sirna, by Santa Cruz Biotechnology (2 mentions)
Interferin reagent, by Polyplus Transfection (2 mentions)
Jetprime dna transfection reagent, by Polyplus Transfection (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Neontm transfection system, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Metafectene reagent, by Biontex (1 mentions)
Accutase, by PromoCell (1 mentions)
Dmem medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Amaxa 4d nucleofector, by Lonza (1 mentions)
Mem alpha medium, by Thermo Fisher Scientific (1 mentions)
Dmem glutamaxtm 1 medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (1 mentions)
42 protocols using jetpei dna transfection reagent
1
Transfection and Differentiation of Rat and Human Neuronal Cell Lines
2
Plasmid Transfection with jetPEI Reagent
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Plasmid transfection was carried out using jetPEI DNA transfection reagent (Polyplus Transfection, Huntingdon, UK) according to manufacturer’s instructions.
3
Mammalian Cell Culture Protocols
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HEK293T and mammary cells were cultured at 37°C in DMEM containing 1 µg/ml of streptomycin, 100 units/ml of penicillin, and 10% fetal bovine serum (FBS). For treatment with estradiol, cells were grown in the same medium containing phenol red-free DMEM supplemented with 5% charcoal-filtered FBS (i.e., estradiol-stripped medium). All media were obtained from Hyclone Laboratories (USA). Plasmids were transfected to cells by using Lipofectamine with PLUS reagent (Thermo Fisher Scientific), NeonTM Transfection System (Thermo Fisher Scientific), Metafectene reagent (Biontex Laboratories, Germany), or jetPEI™ DNA Transfection Reagent (Polyplus-transfection, France).
4
Transient and Stable Overexpression of Transcription Factors
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A Myc-DDK-tagged ORF clone of MAFG, ELK-1 or ABCA1 and the negative control pCMV6 were used for in transient transfection (OriGene, USA). H23 and A2780 cells were plated onto 60-mm dishes at 6x105 cells/dish and transfected with a negative control, MAFG, ELK-1 or ABCA1 vectors (IDs: RC221486; RC208921 and RC221861) using jet-PEI DNA Transfection Reagent (PolyPlus Transfection, USA). For stable overexpression, lentiviruses carrying ELK-1 cDNA (Applied Biological Materials, Canada) were obtained by cotransfecting 15 μg of the specific lentiviral vector (pGIPZ-nonsilencing or pLenti-GIII-CMV-hELK-1-GFP-2A-Puro) and 5 μg of each packaging vector (pCD-NL-BH and pMD2-VSV-G) in 10 million HEK 293T cells using Lipofectamine 2000 (Invitrogen, USA). Supernatants were taken at 48 hours posttransfection. A2780S cells were plated onto 60-mm dishes at 1x105 cells/dish and transduced with supernatant carrying nonsilencing or ELK-1 lentivirus, and polybrene was added (5 μg/ml).
Transfection efficacy was measured by qRT-PCR, using the sensitive cell line transfected with the negative control as a calibrator. Two independent experiments were performed in quadruplicate.
Transfection efficacy was measured by qRT-PCR, using the sensitive cell line transfected with the negative control as a calibrator. Two independent experiments were performed in quadruplicate.
5
Transfection of HEK293T and PACO2 Cells
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HEK293T cells were maintained in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1× penicillin/streptomycin (Sigma). DNA constructs were transfected into HEK293T cells using JetPEI DNA transfection reagent (Polyplus-transfection). PACO2 cells were cultured as described by Noll et al.,30 (link) and the transfection was carried with the Amaxa 4D-Nucleofector (Lonza) following the guidelines by Lonza. Briefly, 1 × 106 cells prior to transfection were isolated with Accutase (PromoCell) treatment and centrifuged at 200 × g for 5 min at room temperature. The supernatant was discarded, and the cells were re-suspended carefully in 100 μL of room temperature supplemented with Nucleofector solution SF per sample. 2 μg of plasmid DNA was then added to the solution, and the tube was gently flanked. The transfection was achieved by applying the pulse CM-120. After the pulse, 500 μL of pre-warmed media was added to the cuvette, and the cells transferred into a new well of a 12-well plate containing 1 mL of pre-warmed growth medium.
6
Dual-Luciferase Reporter Assays in HeLa Cells
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Reporter assays were performed in HeLa cells using the Dual-Luciferase Reporter Assay System (Promega). Cells were seeded in a 24-well plate and 24 hours later were transfected using jetPEI DNA transfection reagent (Polyplus-transfection). Each well was co-transfected with three types of vectors in a total amount of 1210 ng of DNA: 1) 400 ng of a luciferase reporter vector (pGL3 basic) under the regulation of the examined promoter; 2) A total of 800 ng of expression vector (p3XFlag-CMV-10), either carrying no insert or containing an insert encoding the ORF of Pax6, Pax6ΔPD or A-Mitf, 400 ng of each; 3) 10 ng normalizing vector (pRL-TK). Cells were harvested 48 hours after transfection and luminescence was evaluated. Each treatment was carried out in duplicate, and each assay was repeated at least three times.
7
Transient Transfection of HEK 293 Cells
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HEK 293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum and penicillin/streptomycin. Cells were seeded onto 10-cm dishes and grown to 80% confluence before transfection. Cells were transfected with a 1:1 ratio of rat MOPr conjugated to YFP and arrestin tagged with Rluc or with a 1:1:1 ratio of HA-tagged rat MOPr, Gαi-RlucII, and Gβγ-GFP, 24 h before assay, using jetPEI DNA transfection reagent (Polyplus).
8
Lentiviral Pseudoparticle Production for Genetic Screens
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To generate pseudoparticles for delivery of cDNA library or individual ORFs, 4 × 106 Lenti-X 293T cells (Clontec) in 10-cm dishes were co-transfected with plasmids encoding (1) pLOC proviruses (2) HIV-1 gag-pol/delta neo and (3) VSV-G in a ratio of 1/0.8/0.2, respectively. To generate pseudoparticles for delivery of shRNA library or individual shRNAs, 4 × 106 Lenti-X 293T cells were co-transfected with plasmids encoding (1) pGIPZ proviruses (2) pMD.G and (3) pCMVR8.91 in a ratio of 1/0.5/0.5. A total of 12 μg RNA was transfected using 24 μl jetPEI DNA transfection reagent (Polyplus Transfection, France). Transfections were carried out for 6 h, followed by a medium change to DMEM containing 10% FBS. Culture medium was collected at 48 h and 72 h, pooled, filtered through 0.45 μm filter, and stored at −80°C. Target cells seeded into 6-well plates at a density of 4.5 × 105 cells/well were transduced with pseudoparticles by spinoculation at 1000 x g for 45 min at 37°C in a medium containing 10%FBS, 20 mM HEPES, and 4 μg/ml polybrene.
9
Transfection and Imaging of Cell Lines
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CHO dhFr− cells (CRL-9096, American Type Culture Collection) were cultured in MEM Alpha medium (Gibco, Thermo Fisher Scientific, Waltham, USA), COS-7 (kindly provided by A. Renigunta, Marburg), HeLa (kindly provided by R. Jacob, Marburg), OK (kindly provided by B. Fakler, Freiburg) and MDCK (kindly provided by R. Jacob) cells in DMEM GlutaMAXTM-I medium (Gibco), both supplemented with 10% fetal calf serum, 1% penicillin and 1% streptomycin. Cells were kept at 37°C and 5% CO2. Cells were tested negative for mycoplasm. They were seeded on cover slips (Fig. 1 A–C), glass bottom dishes (Figs 2 A–D,G,H and 4 ), or in glass bottom µ-slide VI0.5 flow chambers (ibidi, Martinsried, Germany; Figs 1 D,E, 2 E,F, 3 , 5 C–K and 6 and Figs S1–S5 ) for imaging experiments and on polystyrene dishes for protein extraction. At 2 days after seeding, they were transfected using JetPEI® DNA Transfection Reagent (CHO dhFr− cells; Polyplus Transfection, Illkirch-Graffenstaden, France), JetPRIME® DNA Transfection Reagent (HeLa cells; Polyplus Transfection) and Lipofectamine 2000 reagent (COS-7, OK and MDCK cells; Invitrogen, Carlsbad, USA). Experiments were performed 24 h (TIRF imaging) or 48 h (protein extraction) post transfection.
10
siRNA-mediated Gene Knockdown Protocol
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