FOXA3-WT cDNA (pCMV6-XL5-FOXA3) and vector (pCMV6-XL5) were obtained from Origene (Rockville, MD, USA). FOXA3 mutants at nucleotides 185 (c.185C>T) and 731 (c.731C>T) were generated by site-directed mutagenesis of the FOXA3-WT plasmid (Origene). 10T1/2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (CellGro, Manassas, VA, USA) supplemented with Penicillin/Streptomycin and 10% fetal bovine serum (Hyclone, Logan, UT, USA). A total of 5.0 × 105 10T1/2 cells were transfected using the Nucleofector 96-well shuttle system (Amaxa Biosystems, Cologne, Germany) with either 1.2 μg of control plasmids or with 1.2 μg FOXA3-WT, FOXA3 c.185C>T or FOXA3 c.731C>T. pmaxGFP plasmid (0.3 μg, Amaxa Biosystems) was co-transfected for transfection normalization. Twenty-four hours after nucleofection, cells were treated with 5 μg ml−1 insulin and with 10 μM troglitazone for additional 3 days before cells were harvested for RNA analysis.
Nucleofector 96 well shuttle system
Manufactured by Lonza
Sourced in Germany
The Nucleofector 96-well Shuttle system is a laboratory equipment designed for efficient nucleic acid (DNA, RNA) transfer into a variety of cell types. It facilitates high-throughput transfection and electroporation in a 96-well format. The system provides a standardized, easy-to-use platform for cell line development and screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Nucleocuvette plate, by Lonza (3 mentions)
Sf buffer, by Lonza (2 mentions)
Alt r cas12 electroporation enhancer, by Integrated DNA Technologies (2 mentions)
20 l sf buffer, by Lonza (2 mentions)
Platinum pfx polymerase, by Thermo Fisher Scientific (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
Dulbecco s modified eagle s medium, by Corning (1 mentions)
Pmaxgfp plasmid, by Lonza (1 mentions)
Pcmv6 xl5, by OriGene (1 mentions)
Electroporation enhancer, by Integrated DNA Technologies (1 mentions)
Stealth rnai, by Thermo Fisher Scientific (1 mentions)
10 protocols using nucleofector 96 well shuttle system
1
FOXA3 Mutant Functional Analysis
2
Macrophage Gene Silencing via Nucleofection
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Macrophage nucleofection with siRNA was carried out based on a previous report (48 (link)). Small interfering RNAs (siRNAs) were manufactured based on target gene sequences by GenePharma (China) (Table S1
3
Genetic Modification of 293T Cells
4
Efficient Cell Electroporation for Gene Editing
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Electroporation was performed using the Lonza™ Nucleofector™ 96-well Shuttle™ System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1× phosphate buffered saline (PBS) and resuspended in 20 µL of solution SF or SE (Lonza). Cell suspensions were combined with RNP complex(es), Alt-R Cas9 or Cpf1 (Cas12a) Electroporation Enhancer (Integrated DNA Technologies) and HDR donor template (if applicable). This mixture was transferred into one well of a Nucleocuvette™ Plate (Lonza) and electroporated using manufacturer’s recommended protocols (except for HEK293, which used protocol 96-DS-150). After nucleofection, 75 µL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 µL was transferred to a 96-well culture plate with 175 µL pre-warmed culture media. Transfection plates were incubated at 37 °C and 5% CO2.
5
Genetic Modification of 293T Cells
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293T cells were transduced at a low MOI (<0.3) with a lentivirus expressing BFP under the control of an EF1α promoter (Addgene #71825) and sorted by flow cytometry to produce a pure population of BFP-expressing cells. One day post-AAV transduction, 2 × 105 cells were resuspended in 20 µl SF buffer (Lonza) and nucleofected with 10 µl sgBFP RNP and 100 pmol of ssODN donor, using program DS-150 on a Nucleofector 96-well Shuttle system (Lonza). Cells were then transferred to a 12-well plate containing pre-warmed medium, and grown for 4 days. BFP and GFP fluorescence were measured by flow cytometry on a BD Fortessa, and the results were analyzed using FlowJo v10 software.
6
High-Efficiency Gene Editing by Electroporation
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Electroporation was performed using the Lonza Nucleofector 96-well Shuttle System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1× PBS and resuspended in 20 μL of solution SF or SE (Lonza). HAP1 experiments used ~350,000 cells per nucleofection, and Jurkat experiments used ~500,000 cells per nucleofection. Then, cell suspensions were combined with an RNP complex. For Cas9, the RNP concentration was 4 μM with 4 μM Alt-R Cas9 Electroporation Enhancer. For Cas12a, the RNP concentration was a suboptimal dose of 0.2 μM with 3 μM Alt-R Cas12 Electroporation Enhancer (Integrated DNA Technologies) to provide a more diverse range of editing frequencies. This mixture was transferred into one well of a Nucleocuvette Plate (Lonza) and electroporated using manufacturer’s recommended protocols. After nucleofection, 75 μL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 μL was transferred to a 96-well culture plate with 175 μL pre-warmed culture media. Transfection plates were incubated at 37°C and 5% CO2.
7
Silencing BCL6 in OCI-Ly1 Cells
8
CRISPR-Mediated Genome Editing in Cell Lines
9
CRISPR-Mediated Genome Editing in Cell Lines
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The day post-AAV transduction 2 × 105 cells were resuspended in 20 µl SF buffer (Lonza) and nucleofected along with 10 µl sgCCR5 or sgCXCR4 RNPs and 100 pmol of ssODN donor, using program FF-120 (for K562 cells) or DS-150 (for 293T cells), respectively, on a Nucleofector 96-well Shuttle system (Lonza). Cells were collected 3d post-nucleofection and their genomic DNA isolated (Qiagen DNeasy kit). The CCR5 and CXCR4 loci were amplified by PCR from 400 ng of genomic DNA using Pfx Platinum Polymerase (Invitrogen). 200 ng of purified PCR product was digested overnight with PciI (NEB), then resolved on a 2% agarose gel and analyzed with ImageQuant software.
10
Optimized Electroporation for CRISPR Genome Editing
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Electroporation was performed using the Lonza™ Nucleofector™ 96-well Shuttle™ System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1X PBS and resuspended in 20 µL of solution SF or SE (Lonza). Then, cell suspensions were combined with an RNP complex. For Cas9, the RNP concentration was 4 µM with 4 µM Alt-R Cas9 Electroporation Enhancer. For Cas12a, the RNP concentration was a suboptimal dose of 0.2 µM with 3 µM Alt-R Cas12 Electroporation Enhancer (Integrated DNA Technologies) to provide a more diverse range of editing frequencies. This mixture was transferred into one well of a Nucleocuvette™ Plate (Lonza) and electroporated using manufacturer's recommended protocols. After nucleofection, 75 µL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 µL was transferred to a 96-well culture plate with 175 µL pre-warmed culture media. Transfection plates were incubated at 37°C and 5% CO2.
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