Lipofectamine® LTX Reagent offers a streamlined protocol—no need to remove transfection complexes or change/add medium following transfection. A simple. Lipofectamine LTX® Reagent is a proprietary, animal-origin free formulation for the or contact Technical Services for other specialized transfection protocols. protocol applicable to Invitrogen products, as set forth below (the “Protocol”). by adding 50 μL of Lipofectamine™ LTX to μL of Opti-MEM® medium.
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Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents.
A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro.
Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays. Primary cells are considered more difficult to transfect than immortalized cell lines, as they are more susceptible to toxic agents and may degrade exogenous nucleic acids in the cytoplasm.
HUVEC have a limited lifespan and a relatively low proliferation rate.
Cells can be gene-modified in vitro and in vivo using physical, viral, or chemical methods. Viral transduction is particularly efficient for gene transfer and is favored for in vivo use.
Here, we describe the comparison of transfection of HUVEC using nine chemical transfection reagents, currently commercially available, to identify the reagent that elicits the highest transfection efficiency without compromising cell viability.
Transfection complexes were formed at room temperature in serum-free medium prior to drop-wise addition to HUVEC, followed by incubation for various periods and replacement with complete medium for 24 or 48 h.
Antibiotics and antifungal agents were not used during transfection procedures. Nine commercially available transfection reagents were tested using a range of DNA: All protocols are per-well of a six-well plate. Cells were incubated for 3 h, after which, the complexes were replaced with complete medium. Medium pprotocol removed from the cells, replaced with complexes, and incubated.
Complexes were incubated for 10 min.
The complexes were added to cells in 2 mL complete medium and incubated. DNA was added to the mixture and the complexes incubated for 15 min. The complexes were added directly to cells in 2 mL complete medium and incubated.
DNA was added to the mixture and incubated for a further 10 min. The complexes were incubated on the cells for 3 h before 1 mL complete medium was added. Mixtures were incubated for 5 min and then combined together for a pdotocol 20 min.
Complexes were added to the cells containing 2 mL complete medium and incubated.
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An equal volume of PLUS reagent was added. The mixture was incubated for 5 min. Lipofectamine LTX was added, and the complexes were allowed to form by incubation for 25 min. Cell medium was replaced with lipoectamine mL Opti-MEM I, to which the mixture was added, and incubated for 4 h, after which the complexes were replaced with complete medium. Dachs, unpublished were used as negative and positive controls, respectively. Transfection efficiencies, according to the proportion of EGFP-expressing cells measured by flow cytometry Fig.
Differences in EGFP expression were dependent mainly protocoo transfection reagent.
The length of incubation prior to analysis made a difference in four of the nine reagents tested, and higher expression levels were detected after 48 h compared with 24 h Fig. DNA ratios were tested, but no clear association with transfection efficiency was detected data not shown.
For three of the reagents Effectene, Escort IV, and ExGenthe final lopofectamine of DNA played a role in transfection efficiency, with more DNA in the mixture associated with increased transfection efficiency results not shown. EGFP expression detection by flow cytometry. Representative traces of HUVEC, incubated for 48 h after transfection before analysis by flow cytometry. The proportion of EGFP-positive cells is presented on the graphs.
Lipofectwmine Lipofectamine 2: C Lipofectamine LTX 3: D Lipofectamine LTX 6. Comparison of nine transfection reagents. Each point represents a different ratio of reagent: Mean transfection efficiency for each reagent is shown horizontal bars. Where a ratio was tested more than once, the mean transfection efficiency is plotted. Flow cytometry black, AAD-positive cells indicated that cells analyzed after 48 h tended to have lipofechamine dead cells compared with cells analyzed after 24 h, e.
Mock transfection data showed no difference in cell death at 24 h versus 48 h, i. Cell viability after transfection. C Lipofectamine LTX 6. The gated region for cell debris or necrosis is indicated, as well as the percentage of dead cells according to this method.
Our study demonstrated that a small selection of commercially available chemical transfection progocol was able to transfer exogenous genes efficiently to primary human cells. The nine compounds tested in this study included activated dendrimers, cationic polymers of linear PEI, lipids and polyamines, nonliposomal lipids, polycationic lipids, and cationic lipids, reflecting the broad categories of chemical transfection reagents available and the intense development in this area.
These reagents show promise, as they are produced easily in large quantities, are used rapidly in high-throughput assays, are noninfectious, and can transfer DNA of various sizes.
Chemical transfection reagents have been shown to reduce growth and viability of cells after transfection, possibly as a result of changes in the strength of the cell membrane. A number of reasons have been considered to explain these differences. Optimal expression of transfected genes in vitro is influenced by many factors, including cell type, passage history, confluence, vector structure, size and purity, promoters, a DNA: However, it was observed in this study that transfection efficiencies appear to vary with some batches of HUVEC, and cells extracted from some cords are able to be transfected more efficiently than those from others.
An electroporation protocol for efficient DNA transfection in PC12 cells.
Other studies have reported differences in cell characteristics between HUVEC from single or multiple-pooled donors, 35 which may explain this variability. Increasing the time to analysis from 24 h to 48 h resulted in an increased proportion of EGFP-positive cells for some reagents but also caused a reduction in cell viability.
Therefore, measurement of EGFP expression by flow cytometry may underestimate the total number of cells that was gene-modified initially. Importantly, dead cells tend to detach from the growth surface and thus, were not analyzed in this study. This led to an underestimation of the total amount of cell death caused by transfection but a lipofectaine accurate representation of the number of live cells and proportion of EGFP-expressing cells left to plate out for subsequent assays.
EGFP gene expression allows easy determination of the proportion of cells that is gene-modified on a single-cell basis, detecting the number of cells expressing EGFP and their level of EGFP expression via flow cytometry.
On the other hand, luciferase activity, detected via conversion of a substrate, resulting in amplified signal, determines the behavior of the entire population, thereby losing information about single cells.
Lipotectamine has been reported that some cationic liposome transfection reagents could lead to autofluorescence in fluorescent microscopy and flow cytometry analysis, 38 but our results for mock transfection using Lipofectamine and Lipofectamine LTX showed no autofluorescence. This study analyzed nine currently available, commercial transfection reagents and showed that cationic lipid reagents were the most efficient in gene-modifying HUVEC.
This project was funded by the Cancer Society of New Zealand. We thank Sigma-Aldrich, Fermentas, and Roche, which kindly supplied free samples of their transfection reagents for us to test. There are no prohocol support or associations that would pose a conflict of interest.
Written, informed consent was obtained from each woman who donated an umbilical cord approved by Canterbury Ethics Committee R.
National Center for Biotechnology InformationU.
Journal List J Biomol Tech v. Hunt1 Margaret J. Currie1 Bridget A. Robinson1 and Gabi U. Author information Copyright and License information Disclaimer. BoxChristchurchNew Zealand Phone: This article has been cited by other articles in PMC. Abstract Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. Burlington, Ontario, Canada 9. Open in a separate window. Footnotes There are no financial support or associations that would pose a conflict of interest.
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Current developments in gene transfection agents. Curr Drug Deliv ; lipofedtamine