LipoD293DNA轉染試劑是脂質體DNA轉運工具的升級版本。我們實驗室使用LipoD293DNA轉染試劑成功轉染了HepG2，LNCaP，CHO及HEK293細胞。
接下來，我們樂意就如何提高轉染效率，降低毒性等方面的小技巧同大家分享。
1、LipoD293/DNA的比率。盡管合適的比率由細胞類型決定，但是使用LipoD293/DNA，3:1的固定比率通常能得到很好的轉染效率。
2、每孔中DNA的含量。對于24孔板來說，我每孔使用的0.2到0.5μgDNA。太多的DNA（例如每孔1.0μg）沒有必要，并且會產生較高的毒性。其他規格的細胞培養器皿，可以根據表面積適當調整DNA含量。
3、DNA及LipoD293的稀釋。一定不要使用含有血清的培養基來稀釋二者。高糖的Plain DMEM培養基是不錯的選擇，但是，高糖并不是很重要。千萬不要使用Opti-MEM（invitrogen生產）！Opti-MEM可以影響轉染復合物的形成。我的同事沒有認識到該點的重要性，錯誤地使用了Opti-MEM，導致轉染效率低下。
4、血清/抗生素的存在與否對轉染沒有影響。目前，我們正在使用的哺乳動物細胞通常是用LipoD293進行轉染的，血清/抗清素對于轉染效率沒有影響。與其他依賴于脂質體的轉染試劑不同，LipoD293不受血清/抗生素的影響，因此您不必擔憂轉染中含血清培養造成的高細胞死亡率問題。
5、轉染后更換培養基，對于我們正在研究的哺乳動物細胞，通常，我們是在轉染后24小時更換培養基，沒有必要在轉染后5小時更換培養基。
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.