Cells were centrifuged at 90g for 10 min, the supernatants were removed, the cell pellets were resuspended in 4D‐Nucleofector™ solution (SF cell line 4D‐Nucleofector X kit L, Lonza, Basel, Switzerland) containing the plasmids and transferred into the Nucleocuvette™ vessels. The vessels were then placed into the retainer of the 4D‐Nucleofector™ X unit and the Nucleofection™ process was ran with program DN‐100. After the run completed, the vessels were removed from the retainer and incubated for 10 min. The cells were resuspended with prewarmed medium and mixed by pipetting for three times, then plated onto cell culture plates for further experiments.
Sf cell line 4d nucleofector x kit l
Manufactured by Lonza
Sourced in Switzerland, United States
The SF Cell Line 4D-Nucleofector X Kit L is a lab equipment product designed for the transfection of a variety of cell lines. It provides the necessary reagents and protocols for the efficient delivery of nucleic acids into cells using the 4D-Nucleofector X System.
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Lab products found in correlation
4d nucleofector, by Lonza (3 mentions)
4d nucleofector system, by Lonza (2 mentions)
4d nucleofection system, by Lonza (2 mentions)
Nucleocuvette, by Lonza (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Natocor (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Geneticin, by InvivoGen (1 mentions)
Amaxa 4d nucleofector, by Lonza (1 mentions)
4d nucleofector x unit, by Lonza (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Torin 1, by Cell Signaling Technology (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Methocult, by STEMCELL (1 mentions)
16 protocols using sf cell line 4d nucleofector x kit l
1
Nucleofection of Cell Lines
2
HLA Coding Sequence Expression in JJN-3 Cells
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HLA coding sequences (IPD accession numbers HLA00401, HLA00404, HLA00413, HLA00420, HLA01451, HLA00427, HLA00433, HLA00434, HLA01672, HLA00446, HLA00455, and HLA00467) were downloaded from IPD-IMGT/HLA (https://www.ebi.ac.uk/ipd/imgt/hla/ ), synthesized (Thermo Fisher Scientific), cloned into pHSE3’ as previously described (27 (link)), and nucleofected (SF Cell Line 4D-Nucleofector X Kit L, Lonza) into 250,000 JJN-3 cells. HLA expression was confirmed by flow cytometry as detailed above.
3
Overexpression of ANGPTL Proteins
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293T cells in T75 flasks were transfected with 10 μg of DNA (ANGPTL3 + empty vector, ANGPTL3 + ANGPTL8, ANGPTL8 + empty vector, or ANGPTL3 + ANGPTL4) and 20 μl of 1 mg/ml PEI (polyethylenimine). HepG2 cells were transfected with the 4D-Nucleofector (Lonza, catalog #AAF-1002B) using the SF Cell Line 4D-Nucleofector® X Kit L (Lonza, catalog #V4XC-2012) following manufacturer's instructions. 24 h post transfection, cells were switched to serum-free DMEM for 293T cells and DMEM/F-12 media (ThermoFisher Scientific, catalog #11320-033) for HepG2 cells. 48–72 h later, conditioned media were collected and concentrated using Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore, catalog #UFC901024). Cells were then washed twice with PBS and lysed with radioimmunoprecipitation assay buffer (1% Nonidet P-40 substitute, 0.5% sodium deoxycholate, and 0.1% SDS in PBS) with protease inhibitor. Cell lysates were cleared by centrifugation at 12,000×g at 4 °C for 5 min. Samples were then analyzed by western blotting.
4
CRISPR/Cas9-Mediated Disruption of S100A9 in RAW264.7 Macrophages
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The murine RAW264.7 macrophage cell line (ATCC TIB-71) and S100A9–/– RAW264.7 were cultured in DMEM (Gibco, United States) supplemented with 10% (v/v) FBS (NATOCOR, Cordoba, Argentina). Briefly, a targeting construct was produced through CRISPR/Cas9 to disrupt S100A9 in the RAW264.7 cell line by Jiangsu Genloci Biotechnologies Inc., China. The complementary oligonucleotide guide RNAs for S100A9 (listed in Supplementary Table S1 ) were annealed and cloned into pGK1.1 (Puro)/CRISPR Cas9. The recombinant plasmids were transfected into RAW264.7 cells using the SF Cell Line 4D-Nucleofector® X Kit L (Lonza, United States). Cell transfection was performed with NEOFECTTM DNA transfection reagent (Neofect Biotech Co., Ltd., Beijing), according to the manufacturer’s instructions. RAW264.7-TNF-α (TNF-α stably expressing cell line) was kindly provided by Professor Zhuoya Li from the Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, China.
5
Silencing S100P in HL-60 Cells
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Control small interfering RNA (siRNA) and S100P targeting siRNA (5′-CAAGGATGCCGTGGATAAA-3′; 5′-GTGTTCGTGGCTGCAATCA-3′; 5′-CGTCTGCCTGTCACAAGTA-3′) specific for human were obtained from RIBOBIO (Guangzhou, China). Transfection of siRNA targeting S100P into the HL-60 cell line was performed on Lonza 4D Nucleofection System (Lonza, Cologne, Germany) to obtain efficient post-transcriptional gene silencing. SF Cell Line 4D-Nucleofector X Kit L was used (Lonza, V4XC-2024) for this experiment. 48 hours after transfection, cells were subjected to subsequent experiments.
6
Overexpression of FLJ23584 in H1975 Cells
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NCI-H1975 WT cells were transfected with a myc-DDK-tagged FLJ23584 ORF clone (RC203355; Origene) using the SF Cell Line 4D-Nucleofector X Kit L (V4XC-2024; Lonza). The same plasmid was used for rescue experiments in non-targeting control and C22orf46 knockout cell lines. For each transfection, one million cells and 5 μg plasmid were used, and the manufacturer’s instructions were followed. 4 d after transfection, the selection with Geneticin (InvivoGen, ant-gn) was started. The cell populations resistant to 600 μg/ml Genetecin were subjected to downstream experiments.
7
Gene Editing in Antibody-Deficient Hybridomas
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Genome editing experiments in B cell hybridomas were always executed in the HC9- cell line being dysfunctional in antibody expression and constitutively expressing Cas9 protein (30 (link)). Hybridoma cells were electroporated using the SF Cell Line 4D-Nucleofector X Kit L (Lonza, V4XC-2024) with the program CQ-104. The following standard conditions in 100 μl of total volume of nucleofection mix were used: 1 × 106 cells, 0.156 nmol pre-complexed Alt-R duplex gRNA and 5 μg of PCR-linearized double-stranded DNA. Following transfection, cells were incubated for 5 min at RT, before adding 500 μl of pre-warmed medium to the nucleocuvette and typically transferring them to 1.5 mL of fresh growth medium in 6-well plates. The cells were usually supplemented 24 h later with 0.5–1.0 mL of fresh culturing medium.
8
Hybridoma Cell Nucleofection and Cloning
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Nucleofection of the HDR template and CRISPR-Cas9 vectors was performed with the SF Cell Line 4D-Nucleofector X Kit L (V4XC-2024, Lonza). Before nucleofection, hybridoma cells were assessed for viability, centrifuged (90g, 5 min), resuspended in phosphate-buffered saline (PBS)/1% FBS, and centrifuged again (90g, 5 min). One million cells were resuspended in SF medium with 1 μg of HDR template and 1 μg of CRISPR-Cas9 vectors or 2 μg of GFP vector (control) and transferred to cuvettes for nucleofection with the 4D-Nucleofection System from Lonza (CQ-104, Program SF). Transfected cells were transferred to a six-well plate in 4 ml of complete medium. The following day, the cells were transferred to a 10-cm petri dish in 10 ml of complete medium supplemented with blasticidin (10 to 20 μg/ml; anti–bl-05, Invivogen). Antibiotic pressure was sustained until GFP-transfected hybridomas were dead and HDR transfections were confluent (typically between days 10 and 14). Subsequently, antibiotic-resistant cells were clonally expanded by seeding the hybridomas in 0.3 cells per well in U-bottom 96-well plates in 100 μl of complete medium. After 10 days, supernatant from wells with a high cell density were obtained for further characterization.
9
Silencing Kindlin-3 in HL-60 Cell Differentiation
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HL-60 cells were maintained in Advanced RPMI containing 10% FBS and 1% pen/strep. HL-60 cells were washed once a day for a minimum of three days to ensure optimal growth. These HL-60s were transfected with control scrambled siRNA or kindlin-3-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by electroporation using the Amaxa Nucleofector 4D using the SF Cell line 4D-nucleofector X Kit L (Lonza, Basel, Switzerland), according to the manufacturer’s instructions and as described previously [4 (link),5 (link)]. HL-60 cells were given fresh media 24 h before electroporation to maximize survival. Cells were pelleted from the media, resuspended in high-resistance nucleofection buffer in the presence of 100 nM siRNA, and electroporated. Immediately after electroporation, cells were transferred to 37 °C media containing 1.3% DMSO and differentiated for 3–5 days under the influence of siRNA prior to experimentation [4 (link),5 (link)]. It was confirmed by Western blot that kindlin-3 expression was silenced up to 90% in dHL-60 cells. Kindlin-3 siRNA-scrambled mutation R556A was used as a control.
10
CRISPR-Edited Single Cell Cloning
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SKM-1 cell line was transfected using 4D-NucleofectorTM X Unit (LONZA) with the standard electroporation program 4D-SF-EH-100 and SF Cell Line 4D-Nucleofector® X Kit L (LONZA) in a 12-well plate. The cells were collected from the culture plate 72 h post-electroporation to be sorted by FACS for single cell cloning and were then subcultured into 96-well plates. After expansion of single cell clone, genotyping of CRISPR target site was performed as previously described.
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