Neuron transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. In a 12-well culture plate, for transfection of cells per well containing 0.5 mL plating medium, 1 μL Lipofectamine 2000 and 1 μg plasmid DNA were first separately diluted in 50 μL minimal essential medium then mixed and incubated at room temperature for 20 min to form the transfection complex. The transfection complex was added to the wells and incubated at 37°C for 4 h before the medium was removed and replaced with feeding medium. Three days following the transfection of neurons, cells were fixed and immunostained or lysed for biochemical analysis. HEK 293T cells transfections were performed at approximately 70% confluency using polyethylenimine reagent (Polysciences, Warrington, PA; cat. # 23966). Two days following the transfection of HEK cells, cells were fixed and immunostained or lysed for biochemical analysis.
Polyethylenimine reagent
Manufactured by Polysciences
Sourced in United States
Polyethylenimine reagent is a cationic polymer used as a transfection agent for the introduction of genetic material into cells. It functions by forming complexes with nucleic acids, facilitating their uptake by cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Luciferase buffer, by Promega (1 mentions)
Lb 9501 luminometer, by Berthold Technologies (1 mentions)
P0448, by Agilent Technologies (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
12 protocols using polyethylenimine reagent
1
Lipofectamine-based Transfection of Neurons and HEK Cells
2
GCaMP5.3 Expression in HEK293T Cells
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HEK293T line cells with 50–70% of confluency were co-transfected with the GCaMP5.3 plasmid using a polyethylenimine reagent (Polysciences Inc., Warrington, PA, USA, #23966) in serum-free Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA, #11058-021). After 3 h of incubation in a CO2 incubator, the Opti-MEM was replaced with full DMEM (Thermo Fisher Scientific, Waltham, MA, USA, #41965-039; 10% FBS; 1% Pen Strep, Thermo Fisher Scientific, Waltham, MA, USA, #15140-122). HEK293T cells were grown to a confluence of 40% to 50% on round coverslips (12 mm diameter) in one well of a 24-well culture plate.
3
Lentivirus Production and Transduction
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To produce lentivirus, the HEK 293T cells seeded in 100-mm plate were transfected with 6.0 μg lentiCXCR4-gRNA-Cas9 or lenti-CRISPR-v2 control plasmids, 4.5 μg psPAX2 and 3.0 μg VSV-G plasmids using Polyethylenimine reagent (PEI, Polysciences, Warrington, PA) according to the manufacturer’s instructions. After incubation for 72 hours, the supernatants of the transfected cells containing lentivirus were harvested and filtered with 0.45 μm filter and then stored at −80 °C. The viral titers were determined by virus counter (Virocyt 2100). The Ghost-CXCR4 cells or Jurkat T cells (1 × 105) seeded in 12-well plate were transduced with the lentivirus at an m.o.i of 40. For the Jurkat T cells, the spin-transduction was performed by centrifuging the plate coated with 8 μg/ml polybrene (Sigma-Aldrich) at 1200 g for 2 hours at 25 °C and cultured for another 2 hours in the incubator. Then, the medium was changed with fresh RPMI 1640 supplemented with 10% FBS. These transduced cells were incubated for 2 days and then collected for genomic DNA extraction or stained with PE-CXCR4 antibody and evaluated by flow cytometry. The HIV-1 strain, NL4-3 (HIV-1NL4-3) was generated as described in our previous work47 (link).
4
Generating Mutant Gαq and Gα16 Constructs
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Mutations of the HA-tagged mouse Gαq cDNA and the human Gα16 cDNA in pcDNA3.1(+) were generated using the QuikChange method with specific primers (Table S3 ) as detailed in the manufacturer's protocol (Stratagene, La Jolla, CA). Successful mutations were verified by DNA sequencing. Subconfluent cell cultures were transiently transfected with the respective expression plasmids using the polyethylenimine reagent (Polysciences, Warrington, PA) following the protocol provided by the manufacturer.
5
Transfection and Lentiviral Infection of Hippocampal Neurons
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Hippocampal neurons were transfected at the time of plating in six-well glass-bottom dishes for FRAP experiments using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA; cat. # 11668019) and the target plasmid DNA or siRNA per the manufacturer’s suggestion. For live imaging experiments, neurons were transfected at DIV5. For one well containing 2 ml plating medium + cells, 3 µl Lipofectamine 2000 and 1 µg plasmid DNA + 2 µl siRNA (20 µm ) were first separately diluted in 50 µl minimal essential medium then mixed and incubated at room temperature for 20 min to form the transfection complex. The transfection complex was added to the wells and incubated at 37°C for 4 h before the medium was removed and replaced with feeding medium. Neurons were then cultured for stated times for FRAP or live imaging experiments.
Recombinant lentiviruses were produced by transfecting HEK293T cells with plasmids for the shRNA constructs with viral packaging and envelope proteins (pRSV/REV, pMDLg/RRE, and pVSVG) using polyethylenimine reagent (Polysciences, Warrington, PA; cat. # 23966). Conditioned medium containing lentivirus was harvested after 48 h, centrifuged at 1000 × g for 10 min, filtered through a 0.45-μm filter, and stored at −80°C. Neurons were infected with lentivirus on the day of plating, and medium was changed 1 d later to feeding medium.
Recombinant lentiviruses were produced by transfecting HEK293T cells with plasmids for the shRNA constructs with viral packaging and envelope proteins (pRSV/REV, pMDLg/RRE, and pVSVG) using polyethylenimine reagent (Polysciences, Warrington, PA; cat. # 23966). Conditioned medium containing lentivirus was harvested after 48 h, centrifuged at 1000 × g for 10 min, filtered through a 0.45-μm filter, and stored at −80°C. Neurons were infected with lentivirus on the day of plating, and medium was changed 1 d later to feeding medium.
6
Generating HA-tagged Mouse Gαq Mutants
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Mutations of the HA-tagged mouse Gαq cDNA, in pcDNA3.1(+), were generated by QuikChange site-directed PCR mutagenesis with specific primers as detailed in Table S1 . Successful mutations were verified by DNA sequencing. Subconfluent cell cultures were transiently transfected with the respective expression plasmids using the polyethylenimine reagent (Polysciences) following the protocol provided by the manufacturer.
7
Flow Cytometric Analysis of GABAA Receptor Expression
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Measurement of surface expression of GABAA receptor β2 subunits using flow cytometry has been described previously.8 (link)
9 (link) Briefly, HEK293T cells were transfected using polyethylenimine reagent (40 kD, Polysciences) at a DNA:transfection reagent ratio of 1:2.5 and harvested 48 hours after transfection. To express wild-type (α1β2γ2s) and mutant (α1β2(T287P)γ2s) receptors, a total of 3 µg of subunit cDNAs were transfected at a ratio of 1:1:1 into 6 cm dishes for most experiments except for whole-cell recording. For mock or single subunit expression, empty pcDNA3.1 vector was added to make a final cDNA transfection amount to 3 μg. The transfected HEK293T cells were removed from the dishes by trypsinisation and then re-suspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline supplemented with 2% fetal bovine serum and 0.05% sodium azide). Following washes with FACS buffer and permeabilisation with Cytofix/cytoperm (BD Biosciences, California, USA) for 15 min, cells were incubated with mouse monoclonal anti-β2/3 antibody (1:200) for 2 hours and then incubated with fluorophore Alexa-647-conjugated goat anti-mouse secondary antibody (1:2000) for 1 hour at 4°C. Cells were then washed with FACS buffer and fixed with 2% paraformaldehyde. The acquired data were analysed using FlowJo 7.1 (Tree Star, Oregon, USA).
9 (link) Briefly, HEK293T cells were transfected using polyethylenimine reagent (40 kD, Polysciences) at a DNA:transfection reagent ratio of 1:2.5 and harvested 48 hours after transfection. To express wild-type (α1β2γ2s) and mutant (α1β2(T287P)γ2s) receptors, a total of 3 µg of subunit cDNAs were transfected at a ratio of 1:1:1 into 6 cm dishes for most experiments except for whole-cell recording. For mock or single subunit expression, empty pcDNA3.1 vector was added to make a final cDNA transfection amount to 3 μg. The transfected HEK293T cells were removed from the dishes by trypsinisation and then re-suspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline supplemented with 2% fetal bovine serum and 0.05% sodium azide). Following washes with FACS buffer and permeabilisation with Cytofix/cytoperm (BD Biosciences, California, USA) for 15 min, cells were incubated with mouse monoclonal anti-β2/3 antibody (1:200) for 2 hours and then incubated with fluorophore Alexa-647-conjugated goat anti-mouse secondary antibody (1:2000) for 1 hour at 4°C. Cells were then washed with FACS buffer and fixed with 2% paraformaldehyde. The acquired data were analysed using FlowJo 7.1 (Tree Star, Oregon, USA).
8
Lentiviral Transduction of Hippocampal Neurons
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HEK293T line cells with 50–70% of confluency were co-transfected with shuttle (lenti-FLAG-EB3, lenti-HA-STIM2, lenti-shRNAi) and two helper plasmids pCMVΔ8.9 and pVSVg using polyethylenimine reagent (Polyscience, # 23966). 48–72 hours after transfection culture medium was collected, centrifuged 5 minutes at 2000 rpm, filtered through 0.45 μm pore, immediately frozen in liquid nitrogen and then stored at −80 °C. Each batch of generated lentiviruses was tested by Western blot in hippocampal neuronal culture infection experiments and the titer with minimal toxicity and maximum infection efficiency was used in all experiments. Lentivirus containing medium volume of 80–200 μl was added per well to WT hippocampal neuronal cultures at DIV8 (next day after calcium phosphate transfection).
9
Transient Transfection and Luciferase Assay
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Transient transfection was performed in dishes of cells with 60~80% confluency using the polyethylenimine reagent (Polysciences, Inc. Warrington, PA) per manufacturer’s protocol. Corresponding empty vectors of expression plasmids were used as substitutions to make equal amounts of total DNA transfected across dishes. Eight hours after transfection, cells were washed with phosphate-buffered saline (PBS), and supplemented with fresh serum-containing media. Cells incubated for 48 h were collected and lysed in luciferase buffer (Promega, Madison, WI). Luciferase activity was quantified using the LB9501 luminometer (Berthold Technologies, Germany) and standardized by total cell protein content as determined by Bradford assay.
10
Detecting CaMKII Isoforms in HEK293T Cells
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HEK293T line cells were co-transfected with Venus-CaMKIIα or GFP-CaMKIIβ with shControl or shCaMKIIβ plasmids using polyethylenimine reagent (Polyscience, #23966) in 1:1 ratio. Co-transfected cells were homogenized in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% NP-40, 1 mM PMSF, protease (Sigma, #S8820) and phosphatase (Sigma, #P0044) inhibitors) on the next day. The total protein lysates were electrophoreticly separated by size on sodium dodecyl sulphate (SDS) polyacrylamide gel. Separated proteins were immobilized to the nitrocellulose membrane. Non-specific proteins on the membrane were blocked by incubation of the membrane in 5% bovine serum albumin (BSA) (Sigma, #A9430) in TBST (tris-buffered saline and Tween 20: 50 мМ Тris, 150мМ NaCl, 0.1% Tween 20, pH = 7.6). Target proteins were detected with anti-green fluorescent protein (1:1000, Invitrogen, #A11122), anti-tubulin (1:1500, DSHB, E7-c) and horseradish peroxidase-conjugated anti-rabbit (1:2000, DAKO, P0448) and anti-mouse (1:2000, DAKO, #P0447) secondary antibodies. Increase of HRP luminescence is achieved using ECL buffer (enhanced chemiluminescence): 9 ml ddH2O, 1.5 ml 1 M Tris-HCl (pH = 8.8), 50 ul luminol [44 mg / ml], 22 ul paracumarate [15 mg / ml] 70 ul 3% H2O2. The enhanced luminescence was recorded on X-ray film.
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