Mouse neutrophil nucleofection was carried out using the 4D-Nucleofector X Unit system (Lonza) following the manufacturer’s instructions. In brief, mouse neutrophils were counted, and 1 × 106 cells were resuspended in 20 μl of Lonza P3 solution with 1 μg of DNA and the solutions were transferred into the X Unit and then subjected to nucleofection in the 4D-Nucleofector using program EA-100. The cells were then resuspended in phenol red–free RPMI (Life Technologies) and seeded into four-chamber 35 mm glass-bottom dishes (No. 1.5 borosilicate coverglass). The cells were incubated at 37 °C in a tissue culture incubator (5% CO2), and used in fluorescence-based assays 3–4 h after transfection.
4d nucleofector x unit system
Manufactured by Lonza
Sourced in Switzerland
The 4D-Nucleofector X Unit system is a compact, high-throughput electroporation device designed for efficient and consistent nucleic acid delivery into a wide range of cell types. It features a fully automated and reproducible electroporation process controlled by the 4D-Nucleofector software.
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Lab products found in correlation
5 protocols using 4d nucleofector x unit system
1
Mouse Neutrophil Nucleofection Procedure
2
Mouse Neutrophil Nucleofection Protocol
3
CRISPR-mediated Gene Editing in rESCs
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rESCs were passaged 48 h before electroporation and cultured in 2i + 10 μM Y-27632. rESC colonies were trypsin dissociated for 10 min followed by serum inactivation. For LTVECs, 4 × 106 cells were electroporated with 2 μg vector using a BTX ECM 630 electroporation system (BTX, Holliston, MA, USA). For CRISPR projects, 2.5 μg of each sgRNA was mixed together and incubated with 20 μg Cas9 protein for 15 min at room temperature. RNPs were added to 2 × 106 rESCs and electroporated using the Lonza 4D-Nucleofector X Unit system (Lonza, Basel, Switzerland). For CRISPR-assisted homology-directed repair (HDR) projects, the RNP/cell mix was combined with 0.4 μg LTVEC prior to nucleofection. After transfection the cells were plated overnight in 2i media, followed by antibiotic resistance selection in one of the following conditions: G418 (75 μg/mL, 3 cycles of 2 days selection +1 day recovery), hygromycin (50 μg/mL for 5 days), or puromycin (0.75 μg/mL for 10 days).
4
Generating Flag-tagged HBx Expressing Plasmids
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Full-length HBx (AF100309.1 ) was inserted into pcDNA3.0 vector to construct HBx plasmids encoding Flag-tagged proteins. The expression plasmid for Flag-tagged HBx has been previously described (Niu et al., 2013 (link)). For electrotransfection, cells were resuspended in PBS buffer at 1.5 × 106 cells per 100 µL, then 3.0 µg plasmid DNA was added. The DNA-cell mixture was transferred to Nucleocuvette™ Vessels and cells were electrotransfected with a Lonza 4D-Nucleofector X-unit system. Preheated medium was added, and the cells were evenly distributed into a 6 cm dish. At 8 h after electrotransfection, medium was replaced with fresh growth medium. Samples were collected within 24 h to extract proteins and nucleic acids.
5
Mouse Neutrophil Nucleofection for Microscopy
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Mouse neutrophil nucleofection was carried out using the 4D-Nucleofector X Unit system (Lonza) following the manufacturer’s instructions. In brief, mouse neutrophils were counted, and 1 × 106 cells were resuspended in 20 μl of Lonza P3 solution with 0.1–1 μg of DNA and the solutions were transferred into the X Unit and then subjected to nucleofection in the 4D-Nucleofector using program EA-100. The cells were then resuspended in phenol red–free RPMI (Life Technologies) and seeded into four-chamber 35-mm glass-bottom dishes (No. 1.5 borosilicate coverglass; In Vitro Scientific). Cells were incubated at 37°C for at least 4 h before microscopy analysis. For the CQ treatment experiments, the transfected cells were left to recover for 1 h after transfections and then incubated in the presence of 50 μM CQ for 4 h before TIRFM analysis.
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