Sf cell line 4d nucleofector x kit

Manufactured by Lonza

Sourced in Germany, Switzerland

The SF Cell Line 4D-Nucleofector X Kit is a laboratory equipment product designed for the transfection of various cell lines. It provides a platform for the efficient delivery of nucleic acids into cells through electroporation technology.

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46 protocols using sf cell line 4d nucleofector x kit

siRNA Knockdown of Ebp1 in 32D Cells

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32D cells were transfected by siEbp1 (the siGENOME SMARTpool, J-00860-05), which purchased from Thermo Fisher Scientific and scrambled control RNA (siCtrl) using electroporation with SF Cell Line 4D-Nucleofector^TM X Kit (Lonza, V4XC-2032) via program CV-137 following the manufacturer’s protocol. The target sequences for siRNAs are shown in Supplementary Table 7.

Zhang L., Nguyen L.X., Chen Y.C., Wu D., Cook G.J., Hoang D.H., Brewer C.J., He X., Dong H., Li S., Li M., Zhao D., Qi J., Hua W.K., Cai Q., Carnahan E., Chen W., Wu X., Swiderski P., Rockne R.C., Kortylewski M., Li L., Zhang B., Marcucci G, & Kuo Y.H. (2021). Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance. Nature Communications, 12, 6154.

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Transfection of Dissociated Sphere Cells

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LN18 spheres cultured for 10 days or WG14 spheres cultured for 21 days were collected by centrifugation, washed with PBS and dissociated to single-cell suspension using TrypLe Express (Thermo Fisher Scientific). 1 × 10⁵ cells were transfected with 50 nM of control or DMRTA2-specific siRNA (Dharmacon) in 20 μl of SF buffer (SF Cell Line 4D-Nucleofector ^TM X Kit) using 4D-Nucleofector system (Lonza). After transfection, cells were immediately resuspended in a fresh medium for spheres without antibiotics, plated on 60 mm plates for cell suspension and incubated for 24 h.

Maleszewska M., Wojnicki K., Mieczkowski J., Król S.K., Jacek K., Śmiech M., Kocyk M., Ciechomska I.A., Bujko M., Siedlecki J., Kotulska K., Grajkowska W., Zawadzka M, & Kaminska B. (2024). DMRTA2 supports glioma stem-cell mediated neovascularization in glioblastoma. Cell Death & Disease, 15(3), 228.

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Culturing CD8+ T Cells under Glucose Conditions

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Raji cells were cultured in RPMI-1640 medium (ThermoFisher Scientific) containing 10% FCS and 1% Penicillin-Streptomycin. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors as described before (24 (link)). Primary human CD8⁺ T cells were negatively isolated from PBMCs using Human CD8⁺ T Cell isolation Kits (Miltenyi Biotec) according to the manufacturer’s instruction. Human CD8⁺ T cells were stimulated with CD3/CD28 activator beads (ThermoFisher Scientific) and cultured in DMEM containing normal (5.6 mM, NG) or high glucose (25 mM, HG) (ThermoFisher Scientific) for 3 days if not otherwise mentioned. Since day 2, additional glucose was added into the medium every two days to compensate the consumed glucose along with recombinant human IL-2 (50 ng/ml, ThermoFisher Scientific). All cells were cultured at 37°C with 5% CO₂. EG7 mouse lymphoma cell line was stably transfected with 4 µg of pCasper-pMax plasmid (25 (link)) using SF Cell Line 4D-NucleofectorTM X Kit (Lonza). 48h after transfection, cells were treated with 0.4 mg/ml G418 and 4 µg/ml puromycin in RPMI-1640 (ThermoFisher Scientific) with 10% FCS. Monoclonally expanded EG7-pCasper cells were cultured in selection medium, supplemented with 1% Penicillin/Streptomycin. Flow cytometry analysis revealed 98% of EG7-pCasper cells were fluorescent.

Zhu J., Yang W., Zhou X., Zöphel D., Soriano-Baguet L., Dolgener D., Carlein C., Hof C., Zhao R., Ye S., Schwarz E.C., Brenner D., Prates Roma L, & Qu B. (2021). High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity. Frontiers in Immunology, 12, 689337.

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Transient Transfection of H9c2 and HL1 Cells

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H9c2 and HL1 cells were cultured up to 80% confluency in antibiotic-free growth media. Before nucleofection, the cells were detached using a Trypsin EDTA solution (Merck, Darmstadt, Germany). The cells (1 × 10⁷ cells/mL) were resuspended in 14.7 µL Nucleofector Solution and 3.3 µL Supplement (SF Cell Line 4D Nucleofector^TM X Kit, Lonza, Basel, Switzerland) with 1 µg pMAXGFP or pMAXGFP-ENPL plasmids (in 2 µL Tris-EDTA buffer). The cells were electroporated by a 4D-Nucleofector instrument (AAF-10002X, Lonza, Basel, Switzerland), using the DS-120 program. After transfection, the cells were plated on glass coverslips (12 mm, VWR, Radnor, PA, USA) coated with 0.02% gelatin (EMD Millipore, St. Louis, MO, USA) and 5 μg/mL fibronectin (Gibco, New York, NY, USA). The efficiency of the transient transfection was examined using a Diaphot TMD Inverted Microscope (Nikon, Tokyo, Japan).

Koncz A., Turiák L., Németh K., Lenzinger D., Bárkai T., Lőrincz P., Zelenyánszki H., Vukman K.V., Buzás E.I, & Visnovitz T. (2023). Endoplasmin Is a Hypoxia-Inducible Endoplasmic Reticulum-Derived Cargo of Extracellular Vesicles Released by Cardiac Cell Lines. Membranes, 13(4), 431.

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Transfection of RAW 264.7 Cells

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Mmu-miR-21a-5p mirVana^® miRNA mimic (4464066), mmu-miR-21a-5p mirVana^® miRNA inhibitor (4464084), and their negative control oligonucleotides (4464076) were purchased from Thermofisher. RAW 264.7 cells were transfected by electroporation using SF Cell Line 4D-NucleofectorTM X Kit (Lonza, Allendale, NJ, USA) and 4DNucleofector^™ X Unit (Lonza, Allendale, NJ, USA), following the manufacturer’s protocol. Transfected cells were cultured for 2 days before further experiments.

Zhou W., Su L., Duan X., Chen X., Hays A., Upadhyayula S., Shivde J., Wang H., Li Y., Huang D, & Liang S. (2018). MicroRNA-21 down-regulates inflammation and inhibits periodontitis. Molecular immunology, 101, 608-614.

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Quantitative Analysis of (P)RR, Globin, ALAS2

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Quantitative real-time RT-PCR of (P)RR, γ-globin, ALAS2 and 18S ribosomal RNA was performed using a 7500 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA), according to the manufacturer's instruction, as previously reported (Kaneko et al. 2009; Kaneko et al. 2012) . The SYBR PrimeScript RT-PCR Kit II (Takara Bio Inc., Shiga, Japan) was used for all reactions. The copy number of (P)RR mRNA, γ-globin mRNA, ALAS2 mRNA or 18S ribosomal RNA was calculated with 7500 system sequence detection software (Applied Biosystems Inc.), using each cDNA as a standard. Relative expression levels of (P)RR mRNA, γ-globin mRNA and ALAS2 mRNA were determined after normalization with 18S ribosomal RNA expression levels.
Transient transfection and small interference RNA (P)RR-specific siRNA was purchased from Invitrogen; ID HSS115474, (siB) sense, 5-AGUUGACCUGCUCUUUCUUUCU GAA-3; anti-sense, 5-UUCAGAAAGA AAGAGCAGGCUAACU-3. RNAi Negative Control Duplexes (Invitrogen) (scramble RNA) was used as a negative control. K562 cells were transfected with 20 nM (P)RR-specific siRNA using SF Cell Line 4D-Nucleofector TM X Kit (Lonza, Belgium), following the manufacturer's instruction. Cells were harvested 48 hours after the transfection, and subjected to realtime RT-PCR of (P)RR mRNA and ALAS2 mRNA, and measurement of cellular heme content.

Kaneko K., Ohba K., Hirose T., Totsune K., Furuyama K, & Takahashi K. (2017). Expression of (Pro)renin Receptor During Rapamycin-Induced Erythropoiesis in K562 Erythroleukemia Cells and Its Possible Dual Actions on Erythropoiesis. The Tohoku journal of experimental medicine, 241(1).

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Generation of Inducible SETD2 KO Cell Lines

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To generate SETD2 KO cell lines (HBL1, OCI-Ly7, SU-DHL-4, SU-DHL-2), parental cells were transduced with lentivirus expressing doxycycline inducible Cas9 and blasticidin resistance gene (Addgene, #83481) followed by 5 days of blasticidin selection. Cas9 expressing cells were transduced with lentivirus expressing a SETD2 specific sgRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’) and GFP (Addgene, #57822). 4 days post doxycycline induction, GFP⁺ cells were single cell sorted into 96 well plates and allowed to grow for at least two weeks. For RIVA, parental cells were electroporated with Amaxa Nucleofector Unit and the SF Cell line 4D-Nucleofector X kit (Lonza, PBC2–22500) to incorporate a recombinant Cas9 nuclease (Alt-R S.p. Cas9 Nuclease V3, Integrated DNA Technologies, 1081058), a SETD2 targeting Alt-R CRISPR-Cas9 crRNA (SETD2G#1: 5’ AAA GAA ACA ATA GTA GAA GT 3’; SETD2G#2: 5’ AAT CTG ATG AAG ATT CTG TA 3’ Integrated DNA Technologies) and Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies, 1075927) using manufacturer’s protocol. Forty-eight hours after electroporation, ATTO550+ single cells were sorted into 96 well plates and allowed to grow for at least two weeks. Clones were screened by PCR amplification of a 500bp region encompassing the CRISPR-Cas9 cleavage site and verified by sanger sequencing at GENEWIZ.

Leung W., Teater M., Durmaz C., Meydan C., Chivu A.G., Chadburn A., Rice E.J., Muley A., Camarillo J.M., Arivalagan J., Li Z., Flowers C.R., Kelleher N.L., Danko C.G., Imielinski M., Dave S.S., Armstrong S.A., Mason C.E, & Melnick A.M. (2022). SETD2 haploinsufficiency enhances germinal center-associated AICDA somatic hypermutation to drive B cell lymphomagenesis. Cancer discovery, 12(7), 1782-1803.

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Neutrophil-like Differentiation and Transfection of HL-60 Cells

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The cell line HL-60 (TCHU 23) was purchased from the Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were plated at 5 × 10⁵ per well cultured in IMDM with 20% FBS combined antibiotics (1:100) according to the recommendation on the official website of ATCC. HL-60 cells were treated with 1.25% DMSO for 4 days to acquire a neutrophil-like phenotype. The DMSO induced HL-60 was an approved model to study biological function of neutrophil as previous article showed59 (link)60 (link)61 (link). Then electrotransfections were exerted through 4D-Nucleofector™ System (Lonza Cologne GmbH 50829 Cologne, Germany) combined with Nucleofector™ Solution under the recommendations of SF Cell Line 4D-Nucleofector™ X Kit. The transfection efficiency was more than 50% which was confirmed through positive plasmid control 0.4 μg pmaxGFP™ Vector and the cell viability (% trypan blue negative cells) is usually around 60 % after 24 hours. The LC3-GFP plasmid was kindly provided by Dr Zhenhong Ni in our department which was generated as described before39 (link). The transfected cells were incubated in humidified 37 °C/5% CO₂ incubator for 24 hours and then were treated for determined conditions and observed under the fluorescence microscope (Olympus IX81, Tokyo, Japan) with 40 lens.

Liu Y.W., Yang T., Zhao L., Ni Z., Yang N., He F, & Dai S.S. (2016). Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome. Scientific Reports, 6, 33614.

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Gene Editing of Lymphoblastic and CD34+ Cells

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Lymphoblastic cell lines from healthy donors or FA‐A patients were transduced with the corresponding IDLV donors [multiplicity of infection (MOI): 50 transducing units/cell (TU/cell)] in 96‐well culture plates in a final volume of 100 μl. One day after transduction, cells were collected, washed, and nucleofected with 1.5–6 μg of total in vitro synthetized mRNA (0.75–3 μg of each ZFN pair), using the SF Cell Line 4D‐Nucleofector X Kit (Lonza).
Purified CD34⁺ cells from healthy donor CBs or from FA‐A patients were prestimulated for 48 or 24 h, respectively, as described above, at a density of 10⁶ cells/ml. Thereafter, 10⁵ cells were transduced with the corresponding IDLV donor at a MOI of 100 TU/cell in a 96‐well culture plate in a final volume of 100 μl. One day after transduction, cells were collected, washed, and nucleofected with 6 μg of total in vitro synthetized mRNA (3 μg of each ZFN pair) using the P3 Primary Cell 4D‐Nucleofector X Kit (Lonza). One day after nucleofection, gene‐edited cells were assessed either in clonogenic assays or in transplantation experiments.
In some experiments, cells nucleofected with the PGK‐FANCA/Puro^R donor IDLV were selected with 0.5 μg/ml of puromycin for 48 h. Cell viability was analyzed by flow cytometry as previously established for MMC survival assay.

Diez B., Genovese P., Roman‐Rodriguez F.J., Alvarez L., Schiroli G., Ugalde L., Rodriguez‐Perales S., Sevilla J., Diaz de Heredia C., Holmes M.C., Lombardo A., Naldini L., Bueren J.A, & Rio P. (2017). Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients. EMBO Molecular Medicine, 9(11), 1574-1588.

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Establishing Stable N2A Cell Lines via DdCBE Nucleofection

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N2A cells (ATCC, CCL-131) were maintained in DMEM supplemented with 10% FBS (Gemini) at 37°C with 5% CO₂, and detected without mycoplasma contamination by PCR test; 400 ng of the DdCBE pair was nucleofected with 4D-Nucleofector (Lonza) according to the manufacturer's protocol using SF Cell Line 4D-Nucleofector X Kit. The nucleofected cells were seeded onto the 12-well plate and supplemented with 2 μg/mL puromycin 24 h post nucleofection; 96 h later, cells were collected for DNA extraction.

Guo J., Chen X., Liu Z., Sun H., Zhou Y., Dai Y., Ma Y., He L., Qian X., Wang J., Zhang J., Zhu Y., Zhang J., Shen B, & Zhou F. (2021). DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome. Molecular Therapy. Nucleic Acids, 27, 73-80.

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