Maess Marten B, Buers Insa, Robenek Horst, Lorkowski Stefan
Institute of Nutrition, Friedrich Schiller University Jena, 07743 Jena, Germany.
Cold Spring Harb Protoc. 2011 May 1;2011(5):pdb.prot5612. doi: 10.1101/pdb.prot5612.
The human monocytic leukemia cell line THP-1 is a widely used model for investigating monocyte and macrophage biology. Successful transfection of THP-1 monocytes with subsequent phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages is not a trivial matter, because according to previous transfection protocols, cell viability is lost almost completely within 24 h of PMA treatment following transfection. This protocol constitutes an optimized version of a previously published protocol by our group. It describes a procedure for transfecting premature THP-1 macrophages, which subsequently can be further differentiated into mature macrophages by PMA without a loss of cell viability. Transfection of THP-1 cells with plasmids or small interfering RNA (siRNA) is achieved by electroporation using the Lonza Nucleofector technology (Basel, Switzerland). This technique allows for the efficient nonviral delivery of plasmids, DNA, RNA, or siRNA into primary cells or cell lines even if the cells are not or are only slowly proliferating. Such cells are usually rather difficult to transfect by nonviral approaches. This means that only viral approaches would be left, which are expensive and labor-intensive and require laboratories complying with the respective safety regulations. The protocol described here is an efficient and convenient alternative.
人单核细胞白血病细胞系THP-1是研究单核细胞和巨噬细胞生物学的一种广泛使用的模型。用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导THP-1单核细胞成功转染并随后分化为巨噬细胞并非易事，因为根据以前的转染方案，转染后经PMA处理24小时内细胞活力几乎完全丧失。本方案是我们小组之前发表的方案的优化版本。它描述了一种转染未成熟THP-1巨噬细胞的程序，随后这些细胞可通过PMA进一步分化为成熟巨噬细胞，而不会丧失细胞活力。使用瑞士巴塞尔的Lonza Nucleofector技术通过电穿孔法将质粒或小干扰RNA（siRNA）转染到THP-1细胞中。即使细胞不增殖或增殖缓慢，该技术也能将质粒、DNA、RNA或siRNA高效地非病毒递送至原代细胞或细胞系中。这类细胞通常很难通过非病毒方法进行转染。这意味着只剩下病毒方法，而病毒方法既昂贵又费力，并且要求实验室遵守各自的安全规定。此处所述的方案是一种高效且便捷的替代方法。
