Often neglected points for optimizing LipoD293 reagent

LipoD293 DNA Transfection Reagent is an enhanced liposome DNA delivery tool. LipoD293 DNA transfection reagent has been used for transfecting HepG2, LNCaP, CHO, and HEK293 cells and has proven to be successful in all cases.
These are some technical points which we think are important to consider in order to achieve maximum efficiency and minimal toxicity while using LipoD293 reagent:

1. Ratio of LipoD293 reagent to DNA: While the optimal ratio is cell type dependent, the ratio of reagent to DNA at 3:1 often gives excellent efficiency. Our lab has been transfecting HepG2, LNCaP, CHO, and HEK293 with LipoD293 reagent at 3:1 ratio with high efficiency.

2. DNA amount per well: For a 24-well plate, we used 0.2 to 0.5 µg of DNA per well. Too much DNA (e.g., 1.0 µg per well) is unnecessary and might lead to high cytotoxicity. For other cell culture formats, adjust the DNA amount per the surface area of the culture dish.

3. Diluent for diluting DNA and LipoD293 reagent: Diluent must be serum free. Plain DMEM medium with high glucose is usually functional, though high glucose has not shown to be necessary. It is not recommended to use Opti-MEM (from Invitrogen). If the appropriate diluent is not used and Opti-MEM is misused, it could lead to suboptimal efficiency. According to our initial testing, Opti-MEM may disrupt the formation of the transfection complex.

4. Transfection with serum and antibiotics: For all mammalian cells we are currently using, 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 reagents, LipoD293 reagent is not usually interfered by serum/antibiotics, so it is not necessary to perform transfection in serum-free medium, which otherwise could result in high cell death.

5. Medium change post transfection: We usually change the medium 24 hours after transfection. A medium change 5 hours post-transfection does not seem necessary for any of the mammalian cells we are currently using.