Subconfluent ADSCs were washed with PBS and incubated in trypsin/EDTA, PBS, and collagenase. The cells were resuspended and pelleted at 800g for 5 minutes and then resuspended in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific). Five hundred microliters of cell suspension was transferred to a 0.4-cm electroporation cuvette (Bio-Rad), and 50 μg of each of the indicated plasmids were added. Empty vector (pcDNA3) was used to normalize the total DNA added in all experiments. The cells were electroporated using a Gene Pulser XceII instrument (Bio-Rad) at 0.18 kV and 950 μF, and the cells were allowed to recover for 10 minutes at room temperature. Equivalent amounts of cell suspension and fresh medium were added together and plated according to the intended experiment.
0.4 cm electroporation cuvette
Manufactured by Bio-Rad
Sourced in United States
The 0.4-cm electroporation cuvette is a laboratory equipment designed for electroporation, a technique used to introduce foreign molecules into cells. The cuvette has a 0.4-cm gap between the electrodes, which is a standard size for many electroporation applications.
Automatically generated - may contain errors
Lab products found in correlation
Gene pulser xcell electroporation system, by Bio-Rad (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Gene pulser system, by Bio-Rad (2 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Electroporation buffer, by Bio-Rad (1 mentions)
Gene pulser 2 electroporation system, by Bio-Rad (1 mentions)
Lb 9507, by Berthold Technologies (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica camera (1 mentions)
C0065, by Solarbio (1 mentions)
P1110, by Solarbio (1 mentions)
Gene pulser xcell system, by Bio-Rad (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions)
Biorad gene pulser 2, by Bio-Rad (1 mentions)
Facsaria iiiu cell sorter, by BD (1 mentions)
Amaxa p3 primary cell 4d nucleofector x kit, by Lonza (1 mentions)
Superscript 2 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
11 protocols using 0.4 cm electroporation cuvette
1
Plasmid Transfection of Adipose-Derived Stem Cells
2
Electroporation-based RNA Transfection Protocol
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Cells were trypsinized, resuspended in complete growth medium, and pelleted at 260 × g for 5 min. Cells were then washed once with 5 ml of Opti-MEM (Thermo Fisher Scientific) and resuspended in Opti-MEM at a concentration of 1.5 × 107 cells/ml. We mixed 400 μl of the cell suspension thoroughly with 10 μg of in vitro-transcribed RNA in a 0.4-cm electroporation cuvette (Bio-Rad). Electrical pulse was exerted once at 270 V, 950 μF, and 100 Ω with the Gene Pulser Xcell electroporation system (Bio-Rad). The electroporated cells were immediately transferred into 10 ml of complete growth medium in a 15-ml Falcon tube. The cell suspension was transferred to a 10-cm dish or T75 flask for normal culturing and was usually passaged at a 1:5 ratio every 3 days. All the supernatant, RNA, protein, and cell samples were harvested during each cell passage with consistent amounts.
3
Viral RNA Electroporation in Vero Cells
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Vero cells grown to 80% confluence were trypsinized and washed twice with 1x PBS by centrifugation at 400xg for 6 minutes at room temperature (RT). Cells were resuspended in electroporation buffer (Biorad, Hercules, CA) to a cell density of 10x106 cells/ml. The in vitro transcribed viral RNA (5μg) was taken in a 0.4 cm electroporation cuvette (Biorad), and cell suspension (2x106 cells in 200 μl) was then added. The mixture was subjected to electroporation using Gene Pulser Xcell Electroporation System at 160V (Biorad), according to the manufacturer’s recommendations. The electroporated cells were aspirated gently and transferred to 6-well plates containing 2 ml of fresh EMEM/10% FBS prewarmed to 37°C. After 16 hours, the medium was removed; cells were washed with 1x PBS and replaced with 2 ml of fresh EMEM/2% FBS. Cells were incubated up to 4 days, and as the CPE became evident, supernatants containing virus were harvested, passaged and titrated, and stored as above.
4
Exosomal Delivery of miRNA Antagomirs
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Exosomes were electroporated with miRNAs (Cy5-miR-203a-3p antagomirs, miR-203a-3p antagomirs, or the negative controls) purchased from GenePharma, with target sequences listed in Table S1 . The electroporation was performed at 400 V/125 µF in a 0.4 cm electroporation cuvette (Bio-Rad, USA). The ratio of exosomes to miRNAs was approximately 3 µg of exosomes to 1 pmol of miRNAs. After electroporation, any unloaded miRNAs that were attached to the surface of the exosomes were removed by performing another round of exosome isolation using the ultracentrifugation method. To quantify the amount of miR-203a-3p antagomirs that were successfully loaded into the exosomes, Cy5-tagged antagomirs were used. The fluorescent signal of the Cy5-tagged antagomirs was evaluated using a microplate reader from Thermo Fisher Scientific.
5
Efficient Plasmid Transfection via Electroporation
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A suspension containing 3×l06 cells (in antibiotic-free DMEM containing 10% FBS) and 10 μg of DNA plasmid were placed in a 0.4 cm electroporation cuvette (Bio-Rad). The electroporation was performed at room temperature, 950 μF and 250 V. Afterwards, the cuvette was kept at room temperature for 4 min. The cells were then resuspended and seeded in an 8-wells Lab-Tek using antibiotic-free 10% FBS DMEM. The media was refreshed one day after electroporation. The cells were used for imaging at two and three days post-electroporation.
6
Electroporation of Primary Mouse Thyroid Cells
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Electroporation of primary mouse thyroid cells was performed as previously described29 (link). Briefly, cells were resuspended in 120 µl Dulbecco’s phosphate-buffered saline (DPBS) and transferred to a 0.4-cm electroporation cuvette (Bio-Rad; Munich, Germany). 40 µg DNA was then added to the cuvette and electroporation was performed using a Bio-Rad Gene Pulser II electroporation system with a capacitance extender set on 0.32 kV and 125 µF. 1 ml warm complete culture medium was added immediately after the electric discharge and the cells were seeded on 24-mm round glass coverslips in six-well plates filled with complete culture medium.
7
Electroporation-based Renilla Luciferase Assay
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Huh7.5 cells were washed twice with phosphate-buffered saline (PBS) and resuspended in cytomix solution pH 7.6 [120 mM KCl, 0.15 mM CaCl2,10 mM K2HPO4/KH2PO4 (pH 7.6), 25 mM Hepes, 2 mM EGTA, and 5 mM MgCl2] freshly supplemented with 5 mM glutathione and 2 mM adenosine triphosphate (ATP) to a final concentration of 1.5 × 107 cells/ml. Cell suspension (400 μl) was mixed with 10 μg of in vitro transcript, transferred in a 0.4-cm electroporation cuvette (Bio-Rad), and pulsed at 975 μF and 270 V using a Gene Pulser system (Bio-Rad). Electroporated cells were immediately resuspended in 15 ml of DMEM complete and seeded into one six-well plate in duplicates. At 4, 24, 48, 72, and 96 hours, cells were washed once with PBS and lysed using 250 μl of ice-cold Luciferase Lysis Buffer [25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 15 mM K2PO4, 4 mM EGTA, 10% (v/v) glycerol, and 0.1% Triton X-100] freshly supplemented with 1 mM DTT. Samples were stored at −80°C. Renilla luciferase activity in cell lysates (20 μl) was measured with 100 μl of Luciferase Assay Buffer [25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 15 mM K2PO4, and 4 mM EGTA] in duplicates using the tube luminometer LB9507 (Berthold Technologies). Relative light unit (RLU) values from the 4-hour time point after electroporation served as input control for normalization.
8
Plasmid Transfection and Compound Treatment
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For standard plasmid transfection, the cells were resuspended in serum and antibiotic-free DMEM at suitable densities (to achieve 30–50% density on the next day). Two hundred microliters of cell suspension were mixed with 5 μg GFP-LC3 plasmid (Hanheng, Shanghai, China), then transferred to a 0.4-cm electroporation cuvette (BioRad, Hercules, CA, USA), and electroporated using Gene Pulser Xcell™ electroporation system (BioRad).24 (link) The pulse amplitude of electroporation varied between 80 and 120 V with a pulse duration of 20 ms/transfection. When the electroporation was over, the cells were incubated at room temperature for 5 mins and transferred to a 8-well LAB-Tek confocal CHAMBERCVG (LAB-Tek, 155411, Hatfield, PA, USA)
Forty-eight hours after transfection, cells were treated with 1.87 μL Gef, 34 μL EGCG and combined treatment (1.87 μL Gef +34 μL EGCG) for 48 hrs. Subsequently, cells were fixed with 4% PFA (P1110, Solarbio), permeabilized with Triton-X-100, and nuclei were stained with DAPI (C0065, Solarbio). Treated cells were then examined with a Leica TCS SP5 laser scanning confocal microscope equipped with a 40× objective lens.
Forty-eight hours after transfection, cells were treated with 1.87 μL Gef, 34 μL EGCG and combined treatment (1.87 μL Gef +34 μL EGCG) for 48 hrs. Subsequently, cells were fixed with 4% PFA (P1110, Solarbio), permeabilized with Triton-X-100, and nuclei were stained with DAPI (C0065, Solarbio). Treated cells were then examined with a Leica TCS SP5 laser scanning confocal microscope equipped with a 40× objective lens.
9
Transient Transfection of Kisspeptin Receptor in HEK293 and MCF-7 Cells
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HEK293 (ECACC 85120602) and MCF-7 (ECACC 86012803) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, 2 nM L-glutamine, 0.2 unit/ml penicillin, 0.2 μg/ml streptomycin and were maintained at 37°C in a humidified atmosphere containing 5% (v/v) CO2. Both cell lines were reported to express kisspeptin receptor naturally [39 (link), 40 (link)], but no receptor binding and activity were detected in our cells. HEK293 and MCF-7 cells were transiently transfected with 10 μg cDNA of human kisspeptin receptor by electroporation using the Bio-Rad Gene Pulser Xcell™ system and Bio-Rad 0.4 cm electroporation cuvettes. HEK293 cells were electroporated at 300 volts with a capacitance of 950 μF, while MCF-7 cells were electroporated at 320 volts with a capacitance of 950 μF.
10
CRISPR-Modified Murine Cell Lines
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E14 cells were passaged twice and plated at 8 × 104 cells/cm2 for 24 h prior to transfection. Cells were transfected with a total of 2 µg DNA (i.e. pairs of Cas9-2A-EGFP vectors, each expressing single gRNAs: US1+DS1 or DS2) using the Amaxa P3 Primary cell 4D-Nucleofector X Kit (Lonza) and incubated with supplemented GMEM for 36 h. Cells were then resuspended in Dulbecco’s phosphate buffered saline (DPBS, Sigma), diluted in sterile Baxter water (TPS Healthcare/Baxter), supplemented with 2% FBS and kept at 4 °C. The GFP+ cells were collected with a FACS Aria IIIu cell sorter (BD). To generate single cell clones of CRISPR-modified E14 cells, sorted pools of GFP+ cells were plated on 0.1% gelatin-coated 100 mm culture dishes at a density of 18 cells/cm2 and single colonies picked approximately 2 weeks later. RAW 264.7 cells (5 × 106) were resuspended in 250 µL of culture medium containing 10 µL of DPBS ± 20 µg DNA (as pairs of FIRE-targeting Cas9-2A-EGFP vectors, as specified above) and incubated at RT for 10 min. Electroporation was performed in 0.4 cm electroporation cuvettes (BioRad) using the BioRad Gene Pulser II (BioRad), at 320 V and a capacitance of 950 µF. Cells were cultured for 36 h and GFP+ cells were single-cell sorted into polystyrene flat-bottom 96-well plates, using a FACS Aria IIIu cell sorter (BD).
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