Primary cortical neuronal cells were isolated from rat cerebrum regions on embryonic days 16 to 17 and cultured as previously described [33 (link),34 (link)]. Growing cultured cortical neuronal cells were detached by trypsinization once. In order to begin experiments to observe neuronal process elongation, neurons were once again attached to cultured dishes [3 (link),34 (link)]. Rat cortical neuronal cells were transfected using the ScreenFect A transfection kit, according to the manufacturer’s instructions (Fujifilm, Tokyo, Japan). Neurons have approximately 5% transfection efficiency [33 (link),34 (link)]. Under these conditions, attached cells incorporating trypan blue (Nacalai Tesque, Kyoto, Japan) were estimated to be less than 5% in each experiment.
Screenfect a transfection kit
Manufactured by Fujifilm
Sourced in Japan
ScreenFect A is a transfection kit that enables the delivery of nucleic acids, such as plasmid DNA or RNA, into mammalian cells. It provides an efficient and convenient method for gene expression studies and other cell-based experiments.
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5 protocols using screenfect a transfection kit
1
Isolation and Culture of Rat Cortical Neurons
2
Efficient Transfection of siRNA and Plasmids
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Cells were transfected with the respective synthesized 21-mer siRNAs with dTdT (Fasmac, Kanagawa, Japan) using the ScreenFect siRNA transfection kit (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions. The medium was replaced 4 h after transfection and cells were generally used for experiments 48 h after transfection.
Cells were also transfected with the respective plasmids (pEF1a (pEF-BOS modified type) encoding CasRx and pSINmU6 encoding gRNA or pcDNA3.1-N-EGFP-human ACE2 intracellular domain) using the ScreenFect A transfection kit (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions. The medium was replaced 4 h after transfection and cells were generally used for experiments 48 h after transfection. All key reagents and antibodies used are listed inTable 1 .
Cells were also transfected with the respective plasmids (pEF1a (pEF-BOS modified type) encoding CasRx and pSINmU6 encoding gRNA or pcDNA3.1-N-EGFP-human ACE2 intracellular domain) using the ScreenFect A transfection kit (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions. The medium was replaced 4 h after transfection and cells were generally used for experiments 48 h after transfection. All key reagents and antibodies used are listed in
3
Plasmid Transfection with ScreenFect A
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The ScreenFect A transfection kit (Fujifilm) was used for plasmid transfection in accordance with the manufacturer’s instructions. The medium was replaced 4 h after transfection. Unless otherwise indicated, transfected cells were generally used for cell biological and biochemical experiments at 48 h or more after transfection.
Under these conditions, the estimated percentage of attached cells incorporating trypan blue was less than 7.5% in each experiment.
Under these conditions, the estimated percentage of attached cells incorporating trypan blue was less than 7.5% in each experiment.
4
Transient Transfection of Cells
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Cells were transiently transfected with plasmids and synthesized 21 mer small interfering (si)RNAs with dTdT using the ScreenFect A transfection kit and the ScreenFect siRNA transfection kit (Fujifilm), respectively, in accordance with the manufacturer’s instructions. The medium was replaced 4 h after transfection and was generally used for 48 to 72 h after transfection for cell biological and biochemical experiments. Under these conditions, attached cells incorporating trypan blue were estimated to be less than 5% in each experiment.
5
Transfection of siRNA and CasRx Plasmid
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Cells were transfected with the respective synthesized 21-mer siRNAs with dTdT or plasmids (Addgene Gene No. 237577 plasmid expressing CasRx and pSINmU6 encoding Grna) using the ScreenFect siRNA or ScreenFect A transfection kit (Fujifilm) in accordance with the manufacturer’s instructions, respectively [16 (link),31 (link)]. The medium was replaced 4 h after transfection and generally used for ≥48 h after transfection for biochemical experiments. In these conditions, attached cells incorporating trypan blue (Nacalai Tesque) were estimated to be <5% in each experiment 48 h after transfection [16 (link),31 (link)].
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