The cel-miR-54 (GenePharma, China) sequence is listed in Additional file 1 : Table S2. Exosomes were loaded with cel-miR-54 through electroporation. Briefly, 100 µg exosomes were mixed with 0.5 OD cel-miR-54 mimics in 4 mm electroporation cuvette (BioRad, USA) and electroporated on Gene Pulser XcellTM Total System (BioRad, USA) at 700 V/150 µF. After electroporation, the mixture were placed on ice for at least 30 min to recover the membrane.
Gene pulser xcell total system
Manufactured by Bio-Rad
Sourced in United States
The Gene Pulser Xcell Total System is a versatile electroporation device designed for the efficient transfection of DNA, RNA, or other molecules into a variety of cell types. It provides precise control over electrical parameters, enabling researchers to optimize electroporation conditions for their specific applications.
Automatically generated - may contain errors
Lab products found in correlation
Transit mrna transfection kit, by Mirus Bio (5 mentions)
Gene pulser electroporation buffer, by Bio-Rad (3 mentions)
T7 ribomax express large scale rna production system, by Promega (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Collagen coated plates, by BD (2 mentions)
Cel mir 54, by GenePharma (1 mentions)
4 mm electroporation cuvette, by Bio-Rad (1 mentions)
0.4 cm gap cuvette, by Bio-Rad (1 mentions)
T7 ribomax express, by Promega (1 mentions)
Non targeting pool, by Horizon Discovery (1 mentions)
Gene pulser cuvette, by Bio-Rad (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
On targetplus smartpool, by Horizon Discovery (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Cd cho medium, by Thermo Fisher Scientific (1 mentions)
Octet red96, by Molecular Devices (1 mentions)
Clonepix2, by Molecular Devices (1 mentions)
Cho k1sv cells, by Lonza (1 mentions)
0.4 cm cuvette, by Bio-Rad (1 mentions)
Pcas9 gfp, by Addgene (1 mentions)
21 protocols using gene pulser xcell total system
1
Exosomal Delivery of cel-miR-54
2
Transformation of Wild-type Algae Cells
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Wild-type cells were transformed by electroporation with GeneArt® MAX Efficiency® Transformation Reagent for Algae protocol and reagent (Invitrogen, Carlsbad, CA, USA). In brief, cells were grown to 1 × 106 cells mL−1 in TAP medium as described. Cells were harvested by centrifugation at 2000× g for 5 min and washed twice with transformation reagent. Cell pellet was resuspended in transformation reagent to a final concentration of 2 × 108 cells mL−1. A total of 1 µg of linearized plasmid was incubated with 250 µL of cells for 5 min on ice. The cell–plasmid mix was then transferred into an ice-cold 0.4 cm–gap cuvette (Bio-Rad, Hercules, CA, USA). Electroporation was performed using the Gene Pulser XcellTM Total System (Bio-Rad, Hercules, CA, USA) with the following conditions: Resistance of 800 Ω, capacity of 50 µF, field strength of 1.25 kV cm−1, and pulse duration of 30 ± 2 ms. Cells were recovered for 14–16 h in 10 mL of TAP supplemented with 40 mM sucrose under dim light at 25 °C, with gentle shaking at 50 rpm and then plated on TAP agar plates supplemented with paromomycin. After 7 days of incubation, single green colonies were transferred to liquid medium containing 25 µg mL−1 paromomycin. Stable transformants were obtained after several rounds of selection and subjected to further analyses.
3
HCV Infection and Viral Propagation
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Unless otherwise indicated, HCV infections were initiated via electroporation with genome-length HCV RNA transcribed in vitro from molecular clones pJFH1-QL (17 (link)) or pH77S.3 (45 (link)) or from the corresponding replication-incompetent controls pJFH1/GND (46 (link)) or pH77S/AAG (47 (link)) using the T7 RiboMax Express large-scale RNA production system (Promega, Madison, WI). For electroporation, 10 μg HCV RNA was combined with 5 × 106 cells in a 4-mm electroporation cuvette and pulsed once at 270 V, 950 μF, and ∞ Ω in a Gene Pulser Xcell Total System (Bio-Rad, Hercules, CA). HepG2-HFL cells were inoculated (multiplicity of infection [MOI] of ~0.5) with infectious JFH1-QL virus generated from HCV RNA-transfected Huh7.5 cells (17 (link)).
4
TRPM7 Knockdown in HUVECs by Electroporation
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Due to the difficulty of transfecting DNA or RNA into HUVECs, electroporation was used for introducing siRNA into these cells. The siRNAs transfected were Human TRPM7 (Dharmacon, L-005393-00) and Non-targeting pool (Dharmacon, D-001810-10-05). As in the case of infection by adenovirus, the cells were passaged from a 80 to 90% confluent dish into 100x20 or 60x15 mm cell culture dishes between 16 and 18 hours before the electroporation. At the electroporation time, approximately 120,000 cells were resuspended in Gene Pulser electroporation buffer (Bio-rad, 165–2676) and placed in a Gene Pulser cuvette, 0.2 cm electrode gap (Bio-rad, 165–2082), along with siRNA at 100 nM or 200 nM. The electroporation of HUVECs was then conducted in a Gene Pulser Xcell Total System (Bio-Rad, 165–2660), with a single pulse of a square wave at 150 V for 20 ms. The electroporated cells were plated on a 35x10 mm cell culture dish (Corning, 430165). Experiments were conducted 2 days after electroporation.
5
Establishing Replicon Cell Lines
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Forty-eight hours post-doxycycline treatment, BHK-21-NPDox-ON cells were washed with phosphate-buffered saline (PBS), trypsinized, and resuspended in complete growth medium. Cells were pelleted by centrifugation (1,000 × g for 5 min at 4°C), washed twice with ice-cold DMEM, and resuspended in ice-cold Gene Pulser Electroporation Buffer (Bio-Rad) at 1 × 107 cells/mL. Cells (0.4 mL) were then mixed with 10 μg of replicon RNA and 2 μg NP RNA, placed into 4 mm gap electroporation cuvettes, and electroporated at 270 V, 100 Ω, and 950 μF in a Gene Pulser Xcell Total System (Bio-Rad). To establish stable replicon cells, 200 μg/mL of G418 was added to the media (without doxycycline) between 24 h and 48 h following electroporation, after which culture medium was changed every 2 to 3 days. Three weeks after G418 selection, the resultant foci were counted. All cells were trypsinized and pooled together in a T-75 flask for expansion. Limiting dilution was subsequently performed to derive single cell clones.
6
In Vitro RNA Transfection Protocol
7
In vitro Transcription and Transfection of Viral RNA
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In vitro transcription of HAV or HCV RNA was carried out using T7 RiboMAX™ Express Large Scale RNA Production System (Promega) as per manufacturer’s protocol. Transfection of viral RNA was performed in a Gene Pulser Xcell Total System (Bio-Rad) as previously described33 or using TransIT®-mRNA Transfection Kit (Mirus) for HAV-Luc RNA as described32 (link).
8
Electroporation-Mediated Fungal Transformation
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Aliquots of 100 μl containing 1 × 107 germinated conidia were mixed with 3, 5, or 10 μg of plasmid DNA and kept on ice for 15 min before electroporation. Each transformation mixture was transferred into a 0.2 cm electroporation cuvette and subjected to electroporation using the Gene Pulser Xcell Total System (Bio-Rad Laboratories, USA). Each transformation mixture was electroporated by a single exponential pulse ranging between 0.8 and 2.0 kV/cm, whereas capacitance and resistance were kept constant at 25 μF and infinite Ω resistance, respectively. Experiments using capacitances of 10 or 50 μF, or lower resistances, were also performed.
Immediately after pulse, 1 ml of ice-cold CM-sorbitol medium was added to each cuvette. Each mixture was transferred to a sterile tube. Then, 1 ml of CM-sorbitol was added again and each mixture was incubated at 15°C for 24 h and 80 r.p.m. for recovery. Finally, each transformation mixture electroporated was poured on Petri dishes exactly as was described for protoplast transformation (see section “Polyethylene Glycol/CaCl2-Mediated Transformation”). Hygromycin-resistant colonies were visible after 15–20 days of incubation at 15°C.
For all the transformation experiments, the same controls described in PEG/CaCl2-mediated transformation were performed. In addition, controls where the pulse was omitted were included.
Immediately after pulse, 1 ml of ice-cold CM-sorbitol medium was added to each cuvette. Each mixture was transferred to a sterile tube. Then, 1 ml of CM-sorbitol was added again and each mixture was incubated at 15°C for 24 h and 80 r.p.m. for recovery. Finally, each transformation mixture electroporated was poured on Petri dishes exactly as was described for protoplast transformation (see section “Polyethylene Glycol/CaCl2-Mediated Transformation”). Hygromycin-resistant colonies were visible after 15–20 days of incubation at 15°C.
For all the transformation experiments, the same controls described in PEG/CaCl2-mediated transformation were performed. In addition, controls where the pulse was omitted were included.
9
In vitro Transcription and Transfection of Viral RNA
10
Huh-7.5 Cell-based HCV Transfection Assay
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At 24 h before transfection, 7.5×104 Huh-7.5 cells were seeded onto a 24-well plate. One day later, media were replaced with fresh media, and the cells transfected with 0.25 μg (per well) HCV RNA encoding GLuc using the TransIT mRNA transfection kit (Mirus) according to the manufacturer’s protocol. After 6 h incubation at 37°C, supernatant fluids were removed for GLuc assay and replaced with fresh media containing compound. Alternatively, 10 μg of HCV RNA was mixed with 5×106 Huh-7.5 cells and electroporated into cells using a Gene Pulser Xcell Total System (Bio-Rad) as described previously52 (link). Transfection of wild-type HCV RNA was performed by electroporating 5 μg HCV RNA in 2.5×106 Huh-7.5 cells and seeded into collagen-coated plates (BD Biosciences). Cells were grown in DMEM supplemented with 25 mM HEPES, 7 ng ml−1 glucagon, 100 nM hydrocortisone, 5 μg ml−1 insulin, 2 mM GlutaMAX, antibiotics, and 2% FBS. Culture supernatants were replaced with the media supplemented with drugs at 6 h and every 48 h thereafter and assayed for GLuc activity.
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