4d nucleofector x

Manufactured by Lonza

Sourced in United States

The 4D-Nucleofector X is a laboratory instrument designed for the efficient transfection of cells. It uses a proprietary Nucleofection technology to deliver nucleic acids, such as DNA or RNA, into a wide range of cell types. The core function of the 4D-Nucleofector X is to facilitate the introduction of genetic material into cells, enabling researchers to study gene function, perform gene editing, or express recombinant proteins.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Glutamax, by Thermo Fisher Scientific (4 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions) Sf k562 or sg raji and ramos buffer, by Lonza (2 mentions) Anti igg fc antibody, by BioLegend (2 mentions) Truecut cas9 protein v2, by Thermo Fisher Scientific (2 mentions) Sf cell line 4d nucleofector kit, by Lonza (2 mentions) Nucleotides, by Merck Group (2 mentions) Chir99021, by Selleck Chemicals (2 mentions) Es dmem medium, by Merck Group (2 mentions) Rpim1640 medium, by Corning (2 mentions) Pd0325901, by Selleck Chemicals (2 mentions) β mercaptoethanol, by Merck Group (2 mentions) Penicillin streptomycin, by Corning (2 mentions) L glutamine, by Corning (2 mentions) Sodium pyruvate, by Corning (2 mentions) Non essential amino acids (neaa), by Corning (2 mentions) Hepes, by Corning (2 mentions) Mel 202, by Merck Group (2 mentions) Sf3b1 k700e and control k562, by Horizon Discovery (2 mentions)

14 protocols using 4d nucleofector x

CRISPR-Cas9 Knockout of DNA Repair Genes in mESCs and CH12F3 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The mESCs were cultured in ES-DMEM medium (Millipore) with 15% Fetal Bovine Serum (FBS, ExCell Bio), Penicillin/Streptomycin (Corning), nucleotides (Millipore), l-glutamine (Corning), nonessential amino acids (Corning), PD0325901 (Selleck), CHIR99021 (Selleck) and LIF (Millipore) at 37°C with 5% CO₂. mESCs in 6-cm dishes were transfected with 7.2 μg pX330-Cas9 plus 1.8 μg GFP expression vector by 4D-nucleofector X (Lonza, solution Cytomix, program GC104), then harvested for genomic DNA 3 days after transfection.
The wild-type, Ku80^–/–, Lig4^–/–, Parp1^–/– and AID^–/– CH12F3 cells were cultured in RPIM1640 medium (Corning) with 15% Fetal Bovine Serum (FBS, ExCell Bio), HEPES (Corning), penicillin–streptomycin (Corning), l-glutamine (Corning), nonessential amino acids (Corning), sodium pyruvate (Corning) and β-mercaptoethanol (Sigma-Aldrich) at 37°C with 5% CO₂. Growing CH12F3 cells were transfected with 1.5 μg pX330-Cas9 or pX330-Cas9-mCherry expression vector per million by 4D-nucleofector X (Lonza, solution M1, procedure DN100) and seeding at 0.5 million cells/ml in fresh medium with 1 μg/ml anti-CD40, 5 ng/ml IL-4 and 0.5 ng/ml TGF-β. After 72 h stimulation, the cells were harvested and genomic DNA was extracted for PEM-seq library construction.

Liu M., Zhang W., Xin C., Yin J., Shang Y., Ai C., Li J., Meng F.L, & Hu J. (2021). Global detection of DNA repair outcomes induced by CRISPR–Cas9. Nucleic Acids Research, 49(15), 8732-8742.

+ Open protocol

+ Expand

Optimized CRISPR-mediated genome editing in mESCs and CH12F3 cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

The mESCs were cultured in ES-DMEM medium (Millipore) with 15% fetal bovine serum (FBS, ExCell Bio), Penicillin/Streptomycin (Corning), Nucleotides (Millipore), L-Glutamine (Corning), Nonessential Amino Acids (Corning), PD0325901 (Selleck), CHIR99021 (Selleck) and LIF (Millipore) at 37°C with 5% CO2. mESCs in 6-cm dishes were transfected with 7.2 μg pX330-Cas9 plus 1.8 μg GFP expression vector by 4D-nucleofector X (Lonza, solution Cytomix, program GC104), then harvested for genomic DNA 3 days after transfection.
The wild-type, Ku80 -/-, Lig4 -/-, Parp1 -/-, and AID -/-CH12F3 cells were cultured in RPIM1640 medium (Corning) with 15% Fetal Bovine Serum (FBS, ExCell Bio), HEPES (Corning), Penicillin-Streptomycin (Corning), L-Glutamine (Corning), Nonessential Amino Acids (Corning), Sodium Pyruvate (Corning) and β-Mercaptoethanol (Sigma-Aldrich) at 37°C with 5% CO2. Growing CH12F3 cells were transfected with 1.5 μg pX330-Cas9 or pX330-Cas9-mCherry expression vector per million by 4D-nucleofector X (Lonza, solution M1, procedure DN100) and seeding at 0.5 million cells/mL in fresh medium with 1μg/mL anti-CD40, 5 ng/mL IL-4, and 0.5 ng/mL TGF-β. After 72 hrs stimulation, the cells were harvested and genomic DNA was extracted for PEM-seq library construction.

Liu M., Zhang W., Xin C., Yin J., Shang Y., Chen A., Li J., Meng F., & Hu J. (2021). Global detection of DNA repair outcomes induced by CRISPR-Cas9.

+ Open protocol

+ Expand

Gene Editing of Immune Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

K562, Raji, and Ramos 2G6 (CRL-1923) cells were obtained from ATCC. Cell lines were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. For gene editing, 2 × 10⁵ cells (K562) or 4 × 10⁵ cells (Raji and Ramos) were pelleted by centrifugation and washed with PBS. AAV6 transductions were performed in 10 μL of serum-free RPMI media for 1 hour at 37°C in a 96-well U-bottom plate prior to electroporation, as previously described.³³ Cas9 RNPs were complexed by mixing 3000 ng TrueCut Cas9 protein v2 (Thermo Fisher) with 60 pmol of gRNA (Synthego) for 15 minutes at room temperature.
Cells were then washed with PBS and resuspended in 20 μL of SF (K562) or SG (Raji and Ramos) buffer (Lonza) and mixed with Cas9 RNPs. If appropriate, cells were also mixed with 1.4–2 μg of plasmid homology donors or 100 pmol phosphorothioate-modified ssODNs as previously described.⁵⁹ Electroporations were performed with the 4D-X Nucleofector (Lonza). K562 cells used pulse code FF-120, Raji cells used pulse code DS-104, and Ramos cells used pulse code CA-137.
J3-, A6-, or GFP-edited Raji and Ramos cells were expanded in culture. J3 and A6-edited cells were stained with anti-IgG-Fc antibody (BioLegend, clone M1310G05). Cells were sorted on a FACSAria II cell sorter based on membrane IgG expression (J3 and A6 editing) or GFP expression.

Rogers G.L., Huang C., Mathur A., Huang X., Chen H.Y., Stanten K., Morales H., Chang C.H., Kezirian E.J, & Cannon P.M. (2023). Reprogramming human B cells with custom heavy chain antibodies. Research Square.

+ Open protocol

+ Expand

Gene Editing in Hematopoietic Cell Lines

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

K562, Raji, and Ramos 2G6 (CRL-1923) cells were obtained from ATCC. Cell lines were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. For gene editing, 2 x 10⁵ cells (K562) or 4 x 10⁵ cells (Raji and Ramos) were pelleted by centrifugation and washed with PBS. AAV6 transductions were performed in 10 μL of serum-free RPMI media for 1 hour at 37°C in a 96-well U-bottom plate prior to electroporation, as previously described.^{33 (link)} Cas9 RNPs were complexed by mixing 3000 ng TrueCut Cas9 protein v2 (Thermo Fisher) with 60 pmol of gRNA (Synthego) for 15 minutes at room temperature.
Cells were then washed with PBS and resuspended in 20 μL of SF (K562) or SG (Raji and Ramos) buffer (Lonza) and mixed with Cas9 RNPs. If appropriate, cells were also mixed with 1.4-2 μg of plasmid homology donors or 100 pmol phosphorothioate-modified ssODNs as previously described.^{59 (link)} Electroporations were performed with the 4D-X Nucleofector (Lonza). K562 cells used pulse code FF-120, Raji cells used pulse code DS-104, and Ramos cells used pulse code CA-137.
J3-, A6-, or GFP-edited Raji and Ramos cells were expanded in culture. J3 and A6-edited cells were stained with anti-IgG-Fc antibody (BioLegend, clone M1310G05). Cells were sorted on a FACSAria II cell sorter based on membrane IgG expression (J3 and A6 editing) or GFP expression.

+ Open protocol

+ Expand

Fluorescent Transfection Techniques for Cell Imaging

Check if the same lab product or an alternative is used in the 5 most similar protocols

HFF transfections were performed with the Lonza 4DX Nucleofector using the P2 Primary Cell Solution kit (PBP2-02250) and the NHDF transfection protocol. Transfections were performed with 1–1.5 μg of pEGFP–N1-α-actinin-1 (a gift from Carol Otey, University of North Carolina, Chapel Hill; Addgene plasmid 11908), 1 μg of pCMV-LifeAct-TagRFP (purchased from Ibidi), 1.4 μg of tdTomato–paxillin-22 (a gift from Michael Davidson, National High Magnetic Field Laboratory and Department of Biological Science, Florida State University; deceased 2015; Addgene 58123), pEGFP-MRLC1 (a gift from Tom Egelhoff, Lerner Research Institute, Cleveland Clinic; Addgene 35680¹), and/or RFP-zyxin (a gift from Anna Huttenlocher, University of Wisconsin–Madison; Addgene plasmid 26720; Bhatt et al., 2002 ).

Owen L.M., Adhikari A.S., Patel M., Grimmer P., Leijnse N., Kim M.C., Notbohm J., Franck C, & Dunn A.R. (2017). A cytoskeletal clutch mediates cellular force transmission in a soft, three-dimensional extracellular matrix. Molecular Biology of the Cell, 28(14), 1959-1974.

+ Open protocol

+ Expand

Efficient AAV Transduction of Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293T cells and HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were seeded overnight to adhere to plates and washed once with PBS prior to AAV transduction in DMEM, with or without FBS or human AB serum. FBS was heat-inactivated at 56°C water bath for 30 min. AAV vectors were added to cells at indicated MOIs, and after 4 h at 37°C, 10% FBS (final volume) was restored to the culture if appropriate.
K562, Raji, and Molt4.8 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were washed twice with PBS, seeded into plates at indicated cell concentrations in RPMI-1640, with or without FBS or human AB serum, and transduced with AAV vectors at indicated MOIs at 37°C. After 2 h (or as indicated), 10% FBS (final volume) was restored to the culture if appropriate. Electroporation of K562 cells was performed using a SF Cell Line 4D-Nucleofector kit and 4D-X Nucleofector using pulse code FF-120 (Lonza, Basel, Switzerland), per the manufacturer’s recommendations. After 2 days, GFP expression was measured by flow cytometry, and vector copy numbers were measured by Droplet Digital PCR (ddPCR) as previously described.³⁶ (link) Detailed protocols for AAV copy number determination are provided in the supplemental methods.

Rogers G.L., Huang C., Clark R.D., Seclén E., Chen H.Y, & Cannon P.M. (2021). Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes. Molecular Therapy. Methods & Clinical Development, 23, 198-209.

+ Open protocol

+ Expand

AAV Transduction of Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK-293T cells and HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were seeded overnight to adhere to plates and washed once with PBS prior to AAV transduction in DMEM, with or without FBS or human AB serum. FBS was heat-inactivated at 56°C water bath for 30 mins. AAV vectors were added to cells at indicated MOIs, and after 4 h at 37°C, 10% FBS (final volume) was restored to the culture if appropriate. K562, Raji, and Molt4.8 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were washed twice with PBS, seeded into plates at indicated cell concentrations in RPMI-1640, with or without FBS or human AB serum, and transduced with AAV vectors at indicated MOIs at 37°C. After 2 h (or as indicated), 10% FBS (final volume) was restored to the culture if appropriate. Electroporation of K562 cells was performed using a SF Cell Line 4D-Nucleofector kit and 4D-X Nucleofector using pulse code FF-120 (Lonza, Basel, Switzerland), per the manufacturer's recommendations. After 2 days, GFP expression was measured by flow cytometry and vector copy numbers were measured by ddPCR as previously described. 39 Detailed protocols for AAV copy number determination are provided in the Supplemental Information.

Rogers G.L., Huang C., Clark R.D.E., Seclén E., Chen H., & Cannon P.M. (2021). Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes.

+ Open protocol

+ Expand

Knockdown and Knockout of TRIM Proteins in A549 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

For siRNA-mediated knockdown (KD), A549 cells in Opti-MEM™ Reduced Serum Medium supplemented with 5% (v/v) FCS were seeded into 24-well tissue culture plates and incubated overnight. Cells were then transfected with 10 nM of siRNAs specific for TRIM16 or with the non-targeting control (NTC) (SiGENOME SMART pool, Dharmacon, Lafayette, CO, USA) using Lipofectamine RNAiMAX (Thermo Fisher Scientific), according to manufacturer’s instructions. At 72 h after siRNA treatment, cells were either harvested for qPCR or infected with different viruses to assess virus growth.
For CRISPR/Cas9 editing, specific guide RNA (sgRNA) for TRIM16, TRIM22, or Scrambled guides were obtained from Synthego. The sgRNA and Cas9 protein (Alt-R^® S.p. Cas9 Nuclease V3, IDT, CAT: 1081059) were then used for genome editing using the Lonza 4D Nucleofection system (Lonza, Singapore), according to manufacturer’s instructions. Cells were transfected using the nucleofector machine (4D-Nucleofector^® X, Lonza) using pre-programmed settings (CM-130) suitable for A549 cells. After nucleofection, cells were seeded into 12-well tissue culture plates and expanded for subsequent experiments. All experiments with TRIM16 KO bulk populations were performed within 10 passages of nucleofection. Single-cell clones were prepared from bulk populations by limiting dilution.

Nigos L.R., Scott N.E., Brooks A.G., Ait-Goughoulte M., Londrigan S.L., Reading P.C, & Farrukee R. (2023). TRIM16 Overexpression in HEK293T Cells Results in Cell Line-Specific Antiviral Activity. Pathogens, 12(6), 852.

+ Open protocol

+ Expand

CRISPR Clover-LMNA HDR Assay Protocol

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

The CRISPR Clover-LMNA HDR assay was adapted from refs. 17 (link) and 18 (link). U2OS cells were seeded and transfected with siCtrl or PALB2 small interfering RNA (siRNA) (Supplementary Information) for a final concentration of 50 nM, using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1 × 10⁶ cells were retransfected with 1 μg of pCR2.1-CloverLMNAdonor, 1 μg pX330-LMNAgRNA1, 1 μg pcDNA3 empty vector or the indicated siRNA-resistant flag-tagged PALB2 construct (Supplementary Information), 0.1 g of piRFP670-N1 (used as transfection control), and 200 pmol of siRNA using the 4D-Nucleofector X (Lonza).^{19 (link)} After 48 hours, cells were replated on glass coverslips, fixed with 4% paraformaldehyde, and expression of mClover-LMNA (indicative of successful HDR) was assayed by fluorescence microscopy. Data represent the mean percentages (± SEM) of mClover-LMNA–positive cells over the iRFP670-positive population from five independent experiments (total n > 500 iRFP-positive cells/condition).

Wiltshire T., Ducy M., Foo T.K., Hu C., Lee K.Y., Belur Nagaraj A., Rodrigue A., Gomes T.T., Simard J., Monteiro A.N., Xia B., Carvalho M.A., Masson J.Y, & Couch F.J. (2019). Functional characterization of 84 PALB2 variants of uncertain significance. Genetics in Medicine, 22(3), 622-632.

+ Open protocol

+ Expand

Targeted Genome Editing in CF2-iPS3 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

CF2-iPS3 cells were co-transfected with the donor DNA plasmid (CF2A or CF2B) in the absence or presence of CRISPR/Cas9n-gRNAs. Briefly, CF2-iPS3 cells were harvested as single cell suspension with Accutase (StemCell Inc.). Then, 1.5 × 10⁶ cells were nucleofected with 2.5 μg of donor DNA plasmid with or without 2.5 μg of each pairs of CRISPR/Cas9n-gRNAs using the 4D Nucleofector X (Lonza) with the P3 Primary Cell solution and Program CA137. Transfected CF2-iPS3 cells were plated in mTeSR1 supplemented with 10 μM Y27632 in Matrigel-coated plate for 24 h post transfection. Two to 3 days after transfection, the culture medium was switched to the selection medium, mTeSR1 containing 0.5–1.0 μg/ml of Puromycin (Sigma Aldrich), and the cells were continuously cultured under Puromycin selection up to 14 days. During the selection, 7–10 days post-transfection, all colonies were manually picked and individually transferred to 24-well plates coated with Matrigel. Genomic DNA was isolated from individual clones and amplified by PCR with primers AP1/AP2, AP3/AP4, and P6r/P7f to screen for successful vector replacement and insertion events, and each PCR product was separated on a 1-2% agarose gel containing ethidium bromide and visualized under UV light.

Suzuki S., Chosa K., Barillà C., Yao M., Zuffardi O., Kai H., Shuto T., Suico M.A., Kan Y.W., Sargent R.G, & Gruenert D.C. (2022). Seamless Gene Correction in the Human Cystic Fibrosis Transmembrane Conductance Regulator Locus by Vector Replacement and Vector Insertion Events. Frontiers in Genome Editing, 4, 843885.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!