Double-stranded RNA oligos representing mature sequences that mimic endogenous miR-342 and miR-363 and a non-specific (NS) miRNA negative control (obtained from IDT, Coralville, Iowa) were electrotransfected at concentrations of 50 or 100 nm into CAG human myeloma cells using program EO-117 on the 4D-Nucleofector system and the Amaxa SF cell line 4D-Nucleofector X Kit (Lonza). Cells were harvested 48 h after transfection for protein and mRNA analysis. For miRNA lentiviral clone transfection, miRNA Lenti-miR vectors (System Biosciences) were used to produce vectors encoding miR-Scramble control or mature miR-342, miR-363, or miR-342+363 under control of the CMV promoter with a GFP reporter to allow monitoring of miRNA-expressing cells. The viruses were packaged in HEK293T cells (ATCC) (22 (link)). 5TGM1 cells were infected at 70% confluency for 48 h with lentiviral particles and polybrene (8 μg/mL). A second infection was repeated for another 48 h. Cells were selected by culturing for 14 days in puromycin (8 μg/mL). Protein and mRNA levels were determined in control and miRNA-overexpressing cells by Western blotting and reverse transcriptase quantitative PCR (RT-qPCR).
Amaxa sf cell line 4d nucleofector x kit
Manufactured by Lonza
The Amaxa SF cell Line 4D-Nucleofector X kit is a laboratory equipment designed for the transfection of various cell types. It enables the delivery of nucleic acids, such as plasmids, siRNA, or mRNA, into cells using an electroporation-based method.
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6 protocols using amaxa sf cell line 4d nucleofector x kit
1
Overexpression of miR-342 and miR-363 in Myeloma Cells
2
Electroporation of K562 cells
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K562 cells (ATCC: CCL-243) were grown in RPMI supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher) in an incubator at 37 °C and 5% CO2 atmosphere. 200,000 cells were electroporated with 1000 ng adRNA plasmid or 48 pmol of IVT RNA using the Amaxa SF cell Line 4D-Nucleofector X kit (Lonza) as per the manufacturer’s instructions.
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Electroporation of K562 cells
4
TP53INP1 Gene Silencing in KPUM‐UH1 Cells
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RNA interference of TP53INP1 was performed by transfecting the shRNA plasmid into KPUM‐UH1 cells. 1.0 × 106 cells were resuspended in 100 μL of transfection buffer and transfected with shRNA plasmid for TP53INP1 (#sc‐76,715‐SH) or with control shRNA plasmid (#sc‐108060) (Santa Cruz Biotechnology, Inc.) using the Amaxa™ SF Cell Line 4D‐Nucleofector™ X Kit (Lonza AG). The shRNA plasmid for TP53INP1 was a pool of three target‐specific plasmids designed to knock down TP53INP1 gene expression.
5
Generation of Stable Lung Cell Lines
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A549 lung epithelial cells were nucleofected with GFP/proSP-C WT , GFP/proSP-C L55F , GFP/proSP-C I73T , or GFP/proSP-C A116D using the Amaxa SF Cell line 4D-Nucleofector X kit (Lonza, Cologne, Germany).
Transfected A549 cells were treated with 600 μg/ml G418 (Roche, Indianapolis, IN) as a neomycin selection marker in a 5% CO2 incubator at 37 °C. Control cells not carrying the resistance marker were used to verify cell death by G418. After 4 wk, several neomycin-resistant clones were selected. Neomycin-resistant cells were assessed and sorted by a BD FACS Aria II cell sorter (Becton-Dickinson, Mountain View, CA) using a 488-nm laser. A549 cells stably transfected with GFP/proSP-C WT , GFP/proSP-C L55F , GFP/proSP-C I73T , or GFP/proSP-C A116D were generated for further analysis.
Transfected A549 cells were treated with 600 μg/ml G418 (Roche, Indianapolis, IN) as a neomycin selection marker in a 5% CO2 incubator at 37 °C. Control cells not carrying the resistance marker were used to verify cell death by G418. After 4 wk, several neomycin-resistant clones were selected. Neomycin-resistant cells were assessed and sorted by a BD FACS Aria II cell sorter (Becton-Dickinson, Mountain View, CA) using a 488-nm laser. A549 cells stably transfected with GFP/proSP-C WT , GFP/proSP-C L55F , GFP/proSP-C I73T , or GFP/proSP-C A116D were generated for further analysis.
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Stable Cell Line Generation for GADD34Δ and K3L
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A stable cell line for endogenous expression of GADD34 Δ1–240 (GADD34Δ) and K3L was generated following the procedure described previously (24 (link)). Using Amaxa SF Cell Line 4D-Nucleofector X Kit (Lonza), WT HEK293T cells were transfected with a transposase-encoding plasmid (pSB100X), and the pSBtet-GADD34Δ/K3L construct (see above). After two days of recovery, nucleofected cells were selected with 300 μg/ml hygromycin (Thermo Scientific). A total of three rounds of selection were performed, each with approximately three doublings. Visual observation of the cell culture monitored the efficiency of the selection. After completion of the selection, cells were regularly maintained with 150 μg/ml hygromycin.
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