Human embryonic kidney cells (HEK293) (ATCC CRL-1573) and PBMCs from AML patient were cultured at 37°C and 5% CO2 in DMEM/F-12 and RPMI 1640 medium (Biochrom, Berlin, Germany), respectively, supplemented with 10% fetal bovine serum. 1×106 of HEK293 cells were nucleofected using Amaxa Cell Line Nucleofector® Kit V (Lonza Group, Walkersville, MD) and 4 μg of plasmid DNA, strictly according to the manufacturer's protocol. The stable cell lines expressing NPM1 variants (NPM1wt, NPM1 R2 and NPM1mut, described fully in following chapter) fused with fluorescent GFP tag were established in the presence of G-418 (400 μg/ml) in the media for two weeks. The patient cells were collected by centrifugation and resuspended at 8 × 106 cells/100 μl for primary AML cells in the Human B Nucleofector® Kit solution (Amaxa Biosystems, Cologne, Germany). PBMCs were nucleofected with 4 μg of appropriate plasmid using the U-013 program of the Nucleofection Device II (Amaxa Biosystems). The nucleofected cells were cultured at 37°C for 1 day and used for immunofluorescence staining (described in Confocal imaging section).
Nucleofection device 2
Manufactured by Lonza
The Nucleofection Device II is a laboratory instrument designed for the transfection of cells. It utilizes an electrical pulse-based method to introduce nucleic acids, such as DNA or RNA, into various cell types, including difficult-to-transfect cells. The device provides a controlled environment for the transfection process and is a tool for researchers in the field of molecular biology and cell biology.
Automatically generated - may contain errors
2 protocols using nucleofection device 2
1
Nucleofection of HEK293 and AML Cells
2
Nucleofection of Human CD3+ T-cells
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Human CD3+ T-cells were resuspended at 5×106 per 100 μL of Nucleofector solution from the human T-cell Nucleofector™ kit at room temperature,5 (link),7 (link) and were nucleofected with 3 μg of MALT1-siRNA or a control SC siRNA using the T-020 program of the Nucleofection Device II (Amaxa Biosystems). Mock-transfected cells nucleofected without siRNA were used as a negative control. After nucleofection, the cells were immediately mixed with 500 μL of prewarmed culture medium and transferred into culture plates. The treated cells were incubated at 37°C and were collected for RNA isolation.5 (link)
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