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Trypsinization-dependent cell labeling with fluorescent nanoparticles.

Serdiuk T, Alekseev S, Lysenko V, Skryshevsky V, Géloën A - Nanoscale Res Lett (2014)

Bottom Line: Trypsin is often used to detach adhered cell subculture from a substrate.However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins.To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Lyon, CarMeN Laboratory, INSA de Lyon, UMR INSERM 1060, Lyon, France ; Institute of High Technologies, Taras Shevchenko National University of Kyiv, 64, Volodymyrska St., 01601 Kyiv, Ukraine.

ABSTRACT
Trypsin is often used to detach adhered cell subculture from a substrate. However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins. The present study shows that cellular uptake of fluorescent nanoparticles is remarkably increased within 24 h after trypsinization. These results highlight the trypsin-induced protein digestion, provoking leaky cell plasma membrane which leads to the strongly enhanced cellular uptake of the nanoparticles. To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization.

No MeSH data available.


Related in: MedlinePlus

Luminosity per one cell as a function of the NP concentrations. NPs have been added for t1 = t0 + 24 h and t2 = t0 + 48 h: green bars - epithelial healthy cells (SG), red bars - epithelial cancer cells (HSC).
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Figure 5: Luminosity per one cell as a function of the NP concentrations. NPs have been added for t1 = t0 + 24 h and t2 = t0 + 48 h: green bars - epithelial healthy cells (SG), red bars - epithelial cancer cells (HSC).

Mentions: CFO NPs have been chosen for our experiments due to a set of their major advantages, such as few-nanometer size, relatively narrow size distribution, sufficiently intense green luminescence under UV excitation, high intracellular uptake without additional chemical functionalization, and natural targeting of cell nuclei. In previous studies[7,11,12], we have always observed, under the same experimental conditions of the present study, an intranuclear localization of NP SiC. Numerous arguments show that the localization of NP SiC is intranuclear. During cell division, DNA is replicated and the nucleus divides. It would be extremely difficult to argue that NPs remain extracellular and follow the changes of the nucleus while remaining fixed on the cell membrane. We also performed confocal microscopy and showed that NP SiC has an intranuclear localization.Figure 2 shows fluorescence images of the 3T3-L1 cells exposed to different concentrations of the CFO NPs, 24 h after the trypsin treatment. One can state complete independence of the cell fluorescence intensity on the NP concentration. Indeed, the fluorescence level is already high at the lowest used NP concentration (0.1 mg/mL).Figure 3 shows the general view of the same cells exposed to increasing concentrations of the CFO NPs, 48 h after the trypsin treatment. A clear dose-response effect can be observed: the higher NP concentration gives the stronger fluorescence of the labeled cells.Figure 4 quantitatively summarizes the observed effects qualitatively illustrated by the previous fluorescence images.The same experiment has also been carried out on two human cell lines: (i) cancer cell line (HSC) and (ii) its healthy equivalent (SG). Figure 5 shows the luminosity per one labeled cell for the case of the human epithelial cells (SG, green bars) and oral squamous carcinoma cell line (HSC, red bars) when the NPs have been added 24 or 48 h after the trypsin treatment, at the same concentrations range used for the 3T3-L1 cells. The insets show typical fluorescence images of the labeled cells for both tested human cell lines: healthy and cancer one.


Trypsinization-dependent cell labeling with fluorescent nanoparticles.

Serdiuk T, Alekseev S, Lysenko V, Skryshevsky V, Géloën A - Nanoscale Res Lett (2014)

Luminosity per one cell as a function of the NP concentrations. NPs have been added for t1 = t0 + 24 h and t2 = t0 + 48 h: green bars - epithelial healthy cells (SG), red bars - epithelial cancer cells (HSC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199777&req=5

Figure 5: Luminosity per one cell as a function of the NP concentrations. NPs have been added for t1 = t0 + 24 h and t2 = t0 + 48 h: green bars - epithelial healthy cells (SG), red bars - epithelial cancer cells (HSC).
Mentions: CFO NPs have been chosen for our experiments due to a set of their major advantages, such as few-nanometer size, relatively narrow size distribution, sufficiently intense green luminescence under UV excitation, high intracellular uptake without additional chemical functionalization, and natural targeting of cell nuclei. In previous studies[7,11,12], we have always observed, under the same experimental conditions of the present study, an intranuclear localization of NP SiC. Numerous arguments show that the localization of NP SiC is intranuclear. During cell division, DNA is replicated and the nucleus divides. It would be extremely difficult to argue that NPs remain extracellular and follow the changes of the nucleus while remaining fixed on the cell membrane. We also performed confocal microscopy and showed that NP SiC has an intranuclear localization.Figure 2 shows fluorescence images of the 3T3-L1 cells exposed to different concentrations of the CFO NPs, 24 h after the trypsin treatment. One can state complete independence of the cell fluorescence intensity on the NP concentration. Indeed, the fluorescence level is already high at the lowest used NP concentration (0.1 mg/mL).Figure 3 shows the general view of the same cells exposed to increasing concentrations of the CFO NPs, 48 h after the trypsin treatment. A clear dose-response effect can be observed: the higher NP concentration gives the stronger fluorescence of the labeled cells.Figure 4 quantitatively summarizes the observed effects qualitatively illustrated by the previous fluorescence images.The same experiment has also been carried out on two human cell lines: (i) cancer cell line (HSC) and (ii) its healthy equivalent (SG). Figure 5 shows the luminosity per one labeled cell for the case of the human epithelial cells (SG, green bars) and oral squamous carcinoma cell line (HSC, red bars) when the NPs have been added 24 or 48 h after the trypsin treatment, at the same concentrations range used for the 3T3-L1 cells. The insets show typical fluorescence images of the labeled cells for both tested human cell lines: healthy and cancer one.

Bottom Line: Trypsin is often used to detach adhered cell subculture from a substrate.However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins.To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Lyon, CarMeN Laboratory, INSA de Lyon, UMR INSERM 1060, Lyon, France ; Institute of High Technologies, Taras Shevchenko National University of Kyiv, 64, Volodymyrska St., 01601 Kyiv, Ukraine.

ABSTRACT
Trypsin is often used to detach adhered cell subculture from a substrate. However, the proteolytic activity of trypsin may harm cells by cleaving the cell membrane proteins. The present study shows that cellular uptake of fluorescent nanoparticles is remarkably increased within 24 h after trypsinization. These results highlight the trypsin-induced protein digestion, provoking leaky cell plasma membrane which leads to the strongly enhanced cellular uptake of the nanoparticles. To prevent this effect, one should expose cells to the nanoparticle (NP)-based fluorescent labels at least 48 h after trypsinization.

No MeSH data available.


Related in: MedlinePlus