4d x nucleofector

Manufactured by Lonza

The 4D-X Nucleofector is a lab equipment designed for transfection of cells. It uses electroporation technology to introduce genetic material into cells.

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Lab products found in correlation

Sf k562 or sg raji and ramos buffer, by Lonza (2 mentions) Truecut cas9 protein v2, by Thermo Fisher Scientific (2 mentions) Sf cell line 4d nucleofector kit, by Lonza (2 mentions) Pcmv lifeact tagrfp, by Ibidi (1 mentions) Rfp zyxin, by Addgene (1 mentions) Pegfp n1 α actinin 1, by Addgene (1 mentions)

Close spelling variants of this product

P3 Primary Cell 4D Nucleofector X Kit for Amaxa 4D device Amaxa SE Cell Line 4D-Nucleofector X Kit SF Cell Line 4D-Nucleofector X Kit S P4 Primary Cell 4D-Nucleofector® X Kit SE Cell Line 4D-Nucleofector X Kit L Primary P2 Cell Line 4D-Nucleofector X Kit 4D-Nucleofector X Unit system P1 Primary Cell 4D-NucleofectorTM X kit P3 Primary Cell 4D-Nucleofector X Kit L SF Cell Line 4D-Nucleofector X Kit L

5 protocols using 4d x nucleofector

Gene Editing of Immune Cell Lines

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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.

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Gene Editing in Hematopoietic Cell Lines

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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.

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Fluorescent Transfection Techniques for Cell Imaging

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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.

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Efficient AAV Transduction of Cell Lines

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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.

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AAV Transduction of Cell Lines

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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.

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