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Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Arsenault J, Cuijpers SA, Niranjan D, Davletov B - J. Cell. Biochem. (2014)

Bottom Line: Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

View Article: PubMed Central - PubMed

Affiliation: MRC-Laboratory of Molecular Biology, Neurobiology Division, Cambridge, CB2 0QH, UK; Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, M5S 3M2.

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Investigating Lipofectamine LTX effects on neuro2A cells. A: CCK-8 assay on Neuro2A cells treated with various doses of Lipofectamine LTX. The consistently used experimental dose is indicated by an arrow. B: Flow cytometry assays show the percentage of morphologically normal cells (left panel), the amount of Annexin V-FITC labeling (middle), and propidium iodide labeling (right). C,D: Live imaging of Neuro2A cells treated with or without Lipofectamine LTX and Lysotracker green. C: Normal distribution of acidic organelles in untreated Neuro2A cells at low magnification (top) and high (bottom). D: Altered distribution and intensity of acidic organelles following Lipofectamine LTX treatment shown at low magnification (top) and high (bottom). Lysotracker green strongly labels the area surrounding the plasma membrane. Low magnification white bar: 20 μm. Higher magnification white bar: 10 μm.
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fig04: Investigating Lipofectamine LTX effects on neuro2A cells. A: CCK-8 assay on Neuro2A cells treated with various doses of Lipofectamine LTX. The consistently used experimental dose is indicated by an arrow. B: Flow cytometry assays show the percentage of morphologically normal cells (left panel), the amount of Annexin V-FITC labeling (middle), and propidium iodide labeling (right). C,D: Live imaging of Neuro2A cells treated with or without Lipofectamine LTX and Lysotracker green. C: Normal distribution of acidic organelles in untreated Neuro2A cells at low magnification (top) and high (bottom). D: Altered distribution and intensity of acidic organelles following Lipofectamine LTX treatment shown at low magnification (top) and high (bottom). Lysotracker green strongly labels the area surrounding the plasma membrane. Low magnification white bar: 20 μm. Higher magnification white bar: 10 μm.

Mentions: To ascertain whether lipofectamine LTX could adversely affect Neuro2A cells we performed various survival assays. CCK-8 assay can quickly and efficiently report any cell deaths by monitoring the colorimetric transition of a tetrazolium salt proportional to the amount of viable cells. As can be seen in Figure 4A, the concentration of lipofectamine LTX used (black arrow) did not display any significant loss of viability in Neuro2A cells. Figure 4B shows three readouts obtained from flow cytometry. The left panel shows the total proportion of cells bearing a normal morphology as determined by forward and side scatter. There was no significant disruption in the size and complexity of Neuro2A cells treated with lipofectamine LTX compared to untreated controls. However, a moderate elevation was observed in Annexin V-FITC labeling compared to control (P < 0.05; middle panel) and a slight but non-significant elevation in propidium iodide labeling. If the cytoplasmic membranes were perforated by the reagent there should have been a substantial elevation in propidium iodide labeling. These effects were nevertheless very subtle. As a previous study had shown that the use of bafilomycin A, an endosomal protein pump inhibitor, could prevent the lipofection mediated entry of Botulinum neurotoxins [Kuo et al., 2010] we used lysotracker green to investigate the distribution of acidic organelles in Neuro2A cells. Firstly, there seemed to be a larger overall increase in the total fluorescent signal or total fluorophore uptake in Neuro2A cells that were treated with lipofectamine LTX. Furthermore, the distribution of acidic endosomal compartments were strongly distributed around the plasma membrane indicating an active endocytotic/exocytotic process. Evidently, this active cellular trafficking would undoubtedly contribute to cargo endocytosis and concomitant exocytosis. Unfortunately, due to proprietary concerns we were unable to investigate the individual contribution of the lipofectamine LTX ingredients.


Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Arsenault J, Cuijpers SA, Niranjan D, Davletov B - J. Cell. Biochem. (2014)

Investigating Lipofectamine LTX effects on neuro2A cells. A: CCK-8 assay on Neuro2A cells treated with various doses of Lipofectamine LTX. The consistently used experimental dose is indicated by an arrow. B: Flow cytometry assays show the percentage of morphologically normal cells (left panel), the amount of Annexin V-FITC labeling (middle), and propidium iodide labeling (right). C,D: Live imaging of Neuro2A cells treated with or without Lipofectamine LTX and Lysotracker green. C: Normal distribution of acidic organelles in untreated Neuro2A cells at low magnification (top) and high (bottom). D: Altered distribution and intensity of acidic organelles following Lipofectamine LTX treatment shown at low magnification (top) and high (bottom). Lysotracker green strongly labels the area surrounding the plasma membrane. Low magnification white bar: 20 μm. Higher magnification white bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263260&req=5

fig04: Investigating Lipofectamine LTX effects on neuro2A cells. A: CCK-8 assay on Neuro2A cells treated with various doses of Lipofectamine LTX. The consistently used experimental dose is indicated by an arrow. B: Flow cytometry assays show the percentage of morphologically normal cells (left panel), the amount of Annexin V-FITC labeling (middle), and propidium iodide labeling (right). C,D: Live imaging of Neuro2A cells treated with or without Lipofectamine LTX and Lysotracker green. C: Normal distribution of acidic organelles in untreated Neuro2A cells at low magnification (top) and high (bottom). D: Altered distribution and intensity of acidic organelles following Lipofectamine LTX treatment shown at low magnification (top) and high (bottom). Lysotracker green strongly labels the area surrounding the plasma membrane. Low magnification white bar: 20 μm. Higher magnification white bar: 10 μm.
Mentions: To ascertain whether lipofectamine LTX could adversely affect Neuro2A cells we performed various survival assays. CCK-8 assay can quickly and efficiently report any cell deaths by monitoring the colorimetric transition of a tetrazolium salt proportional to the amount of viable cells. As can be seen in Figure 4A, the concentration of lipofectamine LTX used (black arrow) did not display any significant loss of viability in Neuro2A cells. Figure 4B shows three readouts obtained from flow cytometry. The left panel shows the total proportion of cells bearing a normal morphology as determined by forward and side scatter. There was no significant disruption in the size and complexity of Neuro2A cells treated with lipofectamine LTX compared to untreated controls. However, a moderate elevation was observed in Annexin V-FITC labeling compared to control (P < 0.05; middle panel) and a slight but non-significant elevation in propidium iodide labeling. If the cytoplasmic membranes were perforated by the reagent there should have been a substantial elevation in propidium iodide labeling. These effects were nevertheless very subtle. As a previous study had shown that the use of bafilomycin A, an endosomal protein pump inhibitor, could prevent the lipofection mediated entry of Botulinum neurotoxins [Kuo et al., 2010] we used lysotracker green to investigate the distribution of acidic organelles in Neuro2A cells. Firstly, there seemed to be a larger overall increase in the total fluorescent signal or total fluorophore uptake in Neuro2A cells that were treated with lipofectamine LTX. Furthermore, the distribution of acidic endosomal compartments were strongly distributed around the plasma membrane indicating an active endocytotic/exocytotic process. Evidently, this active cellular trafficking would undoubtedly contribute to cargo endocytosis and concomitant exocytosis. Unfortunately, due to proprietary concerns we were unable to investigate the individual contribution of the lipofectamine LTX ingredients.

Bottom Line: Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible.Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell.This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

View Article: PubMed Central - PubMed

Affiliation: MRC-Laboratory of Molecular Biology, Neurobiology Division, Cambridge, CB2 0QH, UK; Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, M5S 3M2.

Show MeSH
Related in: MedlinePlus