如何优化LipoD293转染试剂？
LipoD293DNA转染试剂是脂质体DNA转运工具的升级版本。我们实验室使用LipoD293DNA转染试剂成功转染了HepG2，LNCaP，CHO及HEK293细胞。
接下来，我们乐意就如何提高转染效率，降低毒性等方面的小技巧同大家分享。
1、LipoD293/DNA的比率。尽管合适的比率由细胞类型决定，但是使用LipoD293/DNA，3:1的固定比率通常能得到很好的转染效率。
2、每孔中DNA的含量。对于24孔板来说，我每孔使用的0.2到0.5µgDNA。太多的DNA（例如每孔1.0µg）没有必要，并且会产生较高的毒性。其他规格的细胞培养器皿，可以根据表面积适当调整DNA含量。
3、DNA及LipoD293的稀释。一定不要使用含有血清的培养基来稀释二者。高糖的Plain DMEM培养基是不错的选择，但是，高糖并不是很重要。千万不要使用Opti-MEM（invitrogen生产）！Opti-MEM可以影响转染复合物的形成。我的同事没有认识到该点的重要性，错误地使用了Opti-MEM，导致转染效率低下。
4、血清/抗生素的存在与否对转染没有影响。目前，我们正在使用的哺乳动物细胞通常是用LipoD293进行转染的，血清/抗清素对于转染效率没有影响。与其他依赖于脂质体的转染试剂不同，LipoD293不受血清/抗生素的影响，因此您不必担忧转染中含血清培养造成的高细胞死亡率问题。
5、转染后更换培养基，对于我们正在研究的哺乳动物细胞，通常，我们是在转染后24小时更换培养基，没有必要在转染后5小时更换培养基。
Some points which are important but ofetn neglected for optimizing LipoD293 reagent.
LipoD293 DNA Transfection Reagent is an enhanced version of liposome
DNA delivery tool. Our lab has been using LipoD293 DNA transfection
reagent for transfecting HepG2, LNCaP, CHO and HEK293 cells with very
good lucks.
Now we like to share some technical points which I think
are important for maximum efficiency and minimal toxicity and often
ignored:
1. Ratio of LipoD293 reagent/DNA. While the optimal ratio is cell type dependent, the ratio of reagent/DNA locked at 3:1 often gives excellent efficiency. Our labs has been transfecting HepG2, LNCaP, CHO and HEK293 with LipoD293 reagent at 3:1 ratio with very good efficiency.
2. DNA amount per well. For 24-well plate, I used 0.2 to 0.5 µg of DNA per well. Too much DNA (eg, 1.0 µg per well) is unnecessary and might lead to high cytotoxicity. For other cell culture format, adjust DNA amount per culture dish's surface area.
3. Diluent for diluting DNA and LipoD293 reagent. Diluent must be serum free. Plain DMEM medium with high glucose is usually very good. High glucose seems not that important. Never use Opti-MEM (from Invitrogen)! This is very important 'cause my colleagues often failed to recognize importance of the diluent and misused Opti-MEM, leading to suboptimal efficiency. Per our initial test, Opti-MEM is able to disrupt formation of transfection complex.
4. Transfection with or without serum/antibiotics. For all mammalian cells we are currently working on, we always perform transfection with LipoD293 reagent in the presence of serum (10% FBS)/antibiotics without compromising any efficiency, thus greatly simplifying our transfection procedures. Unlike other liposome based reagent, LipoD293 reagent is usually NOT interfered by serum/antibiotics, so do not bother to perform transfection in serum-free medium which otherwise results in high cell death.
5. Medium change post transfection. We usually change medium 24 hours after transfection. Medium change 5 hours post transfection is not necessary for all the mammalian cell we are currency using.
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