AAT Bioquest

Transfectamine 5000: an efficient and reliable DNA delivery system


The impact of any protein in cells is widely studied by over expressing a protein of interest and investigating the biomolecular alterations that occur due to the overexpression of that protein. These studies are undertaken to better understand the functionality of vital proteins, such as tumor suppressor proteins or oncogenes. The process of overexpressing proteins is mainly facilitated using a lipid based DNA delivery system which passively delivers DNA into the cells for subsequent translation into protein. In this proposed study, the transfection efficiency of Transfectamine™ 5000, Lipofectamine™ 2000 and Lipofectamine™ 3000 were compared. Comparative analysis revealed a 2-3 fold increase in transfection efficiency with much lower cytotoxicity when using Transfectamine™ 5000 over its counterparts. Additionally, agonist-stimulated calcium responses with Transfectamine™ 5000 transfected cells was much more pronounced than Lipofectamine™ 2000 and Lipofectamine™ 3000 transfected cells.

Keywords: pH indicator, fluorescence, dual excitation, dual emission, cytotoxicity, WST-8™, intracellular pH



Over the past 30 years, DNA delivery has become a powerful tool for understanding gene structure, regulation and function. Traditionally, it is divided into two main systems: viral vector mediated systems and nonviral mediated systems. Viral vector mediated systems are by far the most efficient for both delivery and expression, and thus highly used in the clinical settings. Despite its high efficiency, it presents caveats such as toxicity, production and packaging issues, and high cost.[1, 2] The potential for increased toxicity and immunogenicity of viral vector mediated systems further limits its use in basic research laboratories. For these reasons, nonviral vector mediated systems have become increasingly desirable in both research laboratories and clinical settings.[3]

DNA delivery systems rely on three main functions: DNA condensation, endocytosis and nuclear entry. DNA molecules, which are negatively charged, are complexed with a cationic transfection reaction system. These complexes then get endocytosed by cells, facilitating the entry of DNA through nuclear pores. Once inside the nucleus, the DNA gets translated and converted into its respective protein. There are several methods through which nonviral DNA delivery system function. This includes a mechanical approach such as microinjection or particle bombardment, an electrical approach such as electroporation or a chemical approach such as artificial lipids, DEAE-dextran, proteins, dendrimers and other polymers.[4]

Artificial lipids such as lipofectamines have been widely popular for the transfection of foreign DNA molecules into cells. In this proposed study, transfection efficiency and toxicity comparisons were made between Transfectamine™ 5000, Lipofectamine™ 2000 and Lipofectamine™ 3000.


Materials and Methods

Artificial Lipids

Transfectamine™ 5000 reagent was obtained from AAT Bioquest (Sunnyvale, CA). Lipofectamine™ 2000 and Lipofectamine™ 3000 reagents were obtained from ThermoFisher (Waltham, MA).

Cell culture

HeLa cells were cultured at 37 °C in DMEM containing heat inactivated serum (10% Fetal Bovine Serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin), under a humidified atmosphere containing 5% CO2. CHO-K1 cells were cultured at 37 °C in F12-K Nutrient mixture containing heat inactivated serum (10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin), under a humidified atmosphere containing 5% CO2.

Transfection Protocol

Cells were seeded in 6-well plates 18 hours prior to transfection. At the time of transfection, cells were grown around 90% confluency in the plate. (Note: Higher confluency is recommended.) Cell culture medium was removed and replaced with 2 mL of fresh culture medium. Transfectamine™ 5000-DNA working solution (2.5 µg GFP DNA, 200 µL Serum-free medium, 7.5 µL Transfectamine™ 5000) was added to each well and incubated at room temperature overnight. GFP transfected cells using Lipofectamine™ 2000 and Lipofectamine™ 3000, were prepared using the manufacturer's recommended transfection protocol. This mix was added to cells and incubated overnight. Next day, cells were transferred to fresh cell culture medium and kept for 48 hours. The expression of GFP was recorded after 72 hours post transfection. Images were acquired using FITC filter set on a fluorescence microscope. Flow cytometry was recorded with 488 nm laser excitation and 530/30 nm filter set (FITC channel).

WST-8 viability test

Transfection was performed on cells (as previously described). Viability was determined by adding 10 µL of WST-8™ solution (AAT Bioquest, Sunnyvale, CA) to each well and incubated at 37 °C for 1 hour. Absorbance was measured at 460 nm using absorbance plate reader.

Calcium Kinetic Assay

Cells were plated in 100 µL culture medium in 96-well bottom black plates (Greiner Bio-One, Kremsmunster, Austria) at 50,000 cells per well. The next day, equal volumes of prepared Calbryte™ 520-loading solutions were added to each well and incubated at 37 °C for 1 hour, under a humidified atmosphere containing 5% CO2. Calcium kinetic assays were performed on FlexStation® (Molecular Devices, Sunnyvale, CA) using the built-in liquid handler to add the calcium flux stimulants (Vasopressin) and the kinetic reading mode to capture the changes in fluorescence signal over time. The kinetic calcium assay data were simultaneously collected by SoftMax®Pro (Molecular Devices, Sunnyvale, CA).


Results and Discussion

Increased transfection efficiency with Transfectamine™ 5000

First, we did transfection of green fluorescence protein (GFP) containing plasmid in various cell lines such as HeLa and CHO-K1 cell lines using various treatments including Transfectamine™ 5000 was and compared the transfection efficiency by numerous applications such as flow cytometry and fluorescence microscopy. We observed that cells transfected with Transfectamine™ 5000 have the highest level of GFP expression (Figure-1a). We calculated the transfection efficiency using the flow cytometry and observed significant increase in GFP expressed cells with Transfectamine™ 5000 upon comparison with their counterparts. (Figure-1b and 1c). Overall, we show much improved transfection efficiency with Transfectamine™ 5000.


Comparison of Lipofectamine™ 2000, Lipofectamine™ 3000 and Transfectamine™ 5000 using various platforms. HeLa cells and CHO-K1 cells were transfected with a GFP expressing plasmid using Lipofectamine™ 2000, Lipofectamine™ 3000 and Transfectamine™ 5000 following their corresponding protocols. Fluorescence images were acquired using the FITC filter (a). Flow cytometry was performed on HeLa cells using 530/30 nm filter (b) and Transfection efficiency was measured base on flow cytometry data (c).

Low cytotoxicity with Transfectamine™ 5000

Since viability of the transfected cells has been a huge issue, we next determined viability of the CHO-K1 cells transfected with different reagents in the presence/absence of plasmid via Cell Meter™ Colorimetric WST-8 Cell Quantification Kit (AAT Bioquest, Sunnyvale, CA) (Figure-2a and b). We observed that cells incubated with Transfectamine™ 5000 have very low cytotoxicity when compared with other transfection reagents. The viability of the cells was further down when transfected with DNA using Lipofectamine™ 2000 and Lipofectamine™ 3000 reagents but no major change was observed in viability with Transfectamine™ 5000.


Cytotoxicity assessment of Transfectamine™ 5000. CHO-K1 cells were incubated with or transfection reagent and GFP plasmid (top) or transfection reagent only (bottom). Viability of cells was assessed using WST-8 cell quantification kit.

Application of GPCR activation

We next tested the calcium response with CHO-K1 cells transfected with G-protein Ga16 and G protein coupled receptor V2R construct using various transfection reagents via vasopressin stimulation.[5] Calcium flux analysis was performed using Calbryte™ 520 AM (AAT Bioquest, Sunnyvale CA) with FlexStation3 (Figure-3). We observed significant change in calcium flux in cells transfected with Transfectamine™ 5000 upon comparison with Lipofectamine™ 2000 and Lipofectamine™ 3000, confirming our previous findings that Transfectamine™ 5000 helps achieve maximum transfection efficiency for in vitro studies.

GPCR activation

GPCR activation of G protein coupled receptor V2R construct transfected cells with Vasopressin using FlexStation 3. The promiscuous G-protein Ga16 and G protein coupled receptor V2R construct were co-transfected in CHO-K1 cells following each transfection reagent's protocol. Intracellular calcium flux assay were performed ~36 hours post transfection. Cells were treated with vasopressin to induce rapid calcium flux and were observed using the calcium sensitive fluorescent dye Calbryte™ 520 AM.



In this assay wise letter, for the transfection process, we have addressed two key issues, one is to improve the transfection efficiency and the other is to minimize the cytotoxicity associated with performing transfection process. We have successfully shown that cells transfected with Transfectamine™ 5000 provides much higher transfection efficiency with very little cytotoxicity.



  1. R.G. Crystal, Transfer of Genes to Humans: Early Lessons and Obstacles to Success, Science. 270 (1995) 404. https://doi.org/10.1126/science.270.5235.404.
  2. S.K. Tripathy, H.B. Black, E. Goldwasser, J.M. Leiden, Immune responses to transgene–encoded proteins limit the stability of gene expression after injection of replication–defective adenovirus vectors, Nat. Med. 2 (1996) 545–550. https://doi.org/10.1038/nm0596-545.
  3. M. Ramamoorth, A. Narvekar, Non-Viral Vectors in Gene Therapy- An Overview, J. Clin. Diagn. Res. JCDR. 9 (2015) GE01–GE06. https://doi.org/10.7860/JCDR/2015/10443.5394.